Anti-tcr antibody molecules and uses thereof

ABSTRACT

The disclosure provides antibody molecules that bind to TCR Vβ regions and multispecific molecules comprising said antibody molecules. Additionally, disclosed are nucleic acids encoding the same, methods of producing the aforesaid molecules, pharmaceutical compositions comprising aforesaid molecules, and methods of treating a cancer using the aforesaid molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/256,917 filed on Dec. 29, 2020, which is a U.S. National PhaseApplication under 35 U.S.C. § 371 of International Application No.PCT/US2019/040592 filed on Jul. 3, 2019, which claims the benefit ofU.S. Provisional Application 62/693,653 filed on Jul. 3, 2018, U.S.Provisional Application 62/737,829 filed on Sep. 27, 2018, U.S.Provisional Application 62/788,674 filed on Jan. 4, 2019, and U.S.Provisional Application 62/808,700 flied on Feb. 21, 2019, the entirecontents of each of which are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 16, 2019, isnamed 53676_729_301_SL.txt and is 1,026,852 bytes in size.

BACKGROUND

Current molecules designed to redirect T cells to promote tumor celllysis for cancer immunotherapy typically target the CD3 epsilon (CD3e)subunit of the T cell receptor (TCR). However, there are limitations tothis approach. Previous studies have shown that, e.g., low doses ofanti-CD3e monoclonal antibody (mAb) can cause T cell dysfunction andexert immunosuppressive effects. In addition, anti-CD3e mAbs bind to allT cells and thus activate a large number of T cells. Suchnon-physiological massive activation of T cells by these anti-CD3e mAbscan result in the production of proinflammatory cytokines such asIFN-gamma, IL-1-beta, IL-6, IL-10 and TNF-alpha, causing a “cytokinestorm” known as the cytokine release syndrome (CRS), which is alsoassociated with neurotoxicity (NT). Thus, it might be advantageous todevelop antibodies that avoid or reduce CRS and/or NT.

SUMMARY OF THE INVENTION

Provided herein, in one aspect, is a method of expanding T cells thatexpresses a T cell receptor beta variable region (TCRβV) in a T cellpopulation, the method comprising: contacting the T cell population witha composition comprising a multispecific molecule, wherein themultispecific molecule comprises a first domain that binds to a firsttarget molecule and a second domain that binds to a second targetmolecule, wherein the first target molecule is a TCRβV and the secondtarget molecule is a target molecule on a target cell that is differentfrom the first target molecule, and wherein the first domain contactsthe TCRβV of a T cell receptor (TCR) expressed by the T cells in the Tcell population, thereby expanding the T cells in the T cell population.

In some embodiments, the T cell population is an in vivo T cellpopulation.

In some embodiments, the second domain comprises a tumor-targetingdomain, a cytokine molecule, or a stromal modifying domain.

In some embodiments, the multispecific molecule comprises at least twonon-contiguous polypeptide chains, wherein a first polypeptide chain ofthe at least two non-contiguous polypeptide chains comprises a firstmember of a dimerization module, and a second polypeptide chain of theat least two non-contiguous polypeptide chains comprises a second memberof the dimerization module, wherein the first polypeptide chain and thesecond polypeptide chain form a complex via the first member of thedimerization module and the second member of the dimerization module.

In some embodiments, the first polypeptide chain comprises the firstdomain and the second polypeptide chain comprises the second domain,wherein: (i) the first polypeptide chain comprises the first domainlinked to the first member of the dimerization module, and the secondpolypeptide chain comprises the second domain linked to the secondmember of the dimerization module; (ii) the first polypeptide chaincomprises a first portion of the first domain linked to the first memberof the dimerization module, and the second polypeptide chain comprises afirst portion of the second domain linked to the second member of thedimerization module; wherein the at least two non-contiguous polypeptidechains comprises a third polypeptide chain comprising a second portionof the first domain and a fourth polypeptide chain comprising a secondportion of the second domain; (iii) the first polypeptide chaincomprises a first portion of the first domain linked to the first memberof the dimerization module, and the second polypeptide chain comprisesthe second domain linked to the second member of the dimerizationmodule; wherein the at least two non-contiguous polypeptide chainscomprises a third polypeptide chain comprising a second portion of thefirst domain; or (iv) the first polypeptide chain comprises the firstdomain linked to the first member of the dimerization module, and thesecond polypeptide chain comprises a first portion of the second domainlinked to the second member of the dimerization module; wherein the atleast two non-contiguous polypeptide chains comprises a thirdpolypeptide chain comprising a second portion of the second domain.

In some embodiments, the multispecific molecule further comprises alinker between the first domain and the first member of the dimerizationmodule, a linker between the second domain and the second member of thedimerization module, a linker between the first portion of the firstdomain and the first member of the dimerization module, a linker betweenthe first portion of the second domain and the second member of thedimerization module, a linker between the first member of thedimerization module and the second domain, a linker between the firstmember of the dimerization module and the first portion of the seconddomain or a combination thereof, wherein the linker is selected from acleavable linker, a non-cleavable linker, a peptide linker, a flexiblelinker, a rigid linker, a helical linker, and a non helical linker.

In some embodiments, the first polypeptide chain comprises the firstdomain and the second domain, wherein the first polypeptide chaincomprises: (i) the first domain linked to the first member of thedimerization module linked to the second domain; (ii) a first portion ofthe first domain linked to the first member of the dimerization modulelinked to a first portion of the second domain, wherein the at least twonon-contiguous polypeptide chains comprises a third polypeptide chaincomprising a second portion of the first domain and a fourth polypeptidechain comprising a second portion of the second domain; (iii) a firstportion of the first domain linked to the first member of thedimerization module linked to the second domain, wherein the at leasttwo non-contiguous polypeptide chains comprises a third polypeptidechain comprising a second portion of the first domain; or (iv) the firstdomain linked to the first member of the dimerization module linked to afirst portion of the second domain, wherein the at least twonon-contiguous polypeptide chains comprises a third polypeptide chaincomprising a second portion of the second domain.

In some embodiments, the multispecific molecule further comprises alinker between the first domain and the first member of the dimerizationmodule, a linker between the second domain and the second member of thedimerization module, a linker between the first portion of the firstdomain and the first member of the dimerization module, a linker betweenthe first portion of the second domain and the second member of thedimerization module, a linker between the first member of thedimerization module and the second domain, a linker between the firstmember of the dimerization module and the first portion of the seconddomain or a combination thereof, wherein the linker is selected from acleavable linker, a non-cleavable linker, a peptide linker, a flexiblelinker, a rigid linker, a helical linker, and a non helical linker.

In some embodiments, the multispecific molecule comprises a polypeptidesequence comprising: (i) the first domain linked to the second domain;(ii) a first portion of the first domain linked to a first portion ofthe second domain, wherein the polypeptide sequence further comprises asecond portion of the first domain and a second portion of the seconddomain; (iii) a first portion of the first domain linked to the seconddomain, wherein the polypeptide sequence further comprises a secondportion of the first domain; or (iv) the first domain linked to a firstportion of the second domain, wherein the polypeptide sequence furthercomprises a second portion of the second domain.

In some embodiments, the polypeptide sequence further comprises a linkerbetween the first domain and the second domain, a linker between thefirst portion of the first domain and the first portion of the seconddomain, a linker between the first portion of the first domain and thesecond domain, a linker between the first domain and the first portionof the second domain, or a combination thereof, wherein the linker isselected from a cleavable linker, a non-cleavable linker, a peptidelinker, a flexible linker, a rigid linker, a helical linker, and anon-helical linker.

In some embodiments, the TCRβV is TCRβV1, TCRβV2, TCRβV3, TCRβV4,TCRβV5, TCRβV6, TCRβV7, TCRβV8, TCRβV9, TCRβV10, TCRβV11, TCRβV12,TCRβV19, TCRβV20, TCRβV21, TCRβV23, TCRβV24, TCRβV25, TCRβV26, TCRβV27,TCRβV28, TCRβV29 or TCRβV30.

In some embodiments, the TCRβV is TCRβV2, TCRβV4-1, TCRβV4-2, TCRβV5-1,TCRβV5-5, TCRβV5-6, TCRβV6, TCRβ6-5, TCRβV6-6, TCRβV6-9, TCRβV7-2,TCRβV7-3, TCRβV7-8, TCRβV7-9, TCRβV9, TCRβV10-1, TCRβV10-2, TCRβV10-3,TCRβV11-2, TCRβV12-3, TCRβV12-4, TCRβV12-5, TCRβV19, TCRβV20-1, TCRβV21,TCRβV24-1, TCRβV25-1 or TCRβV28.

In some embodiments, the TCRβV is TCRβV2, TCRβV3-1, TCRβV4-1, TCRβV4-2,TCRβV5-1, TCRβV5-4, TCRβV5-5, TCRβV5-6, TCRβV6-1, TCRβV6-5, TCRβV6-6,TCRβV7-3, TCRβV7-6, TCRβV7-8, TCRβV9, TCRβV11-2, TCRβV19, TCRβV20-1,TCRβV24-1, TCRβV27, TCRβV28, TCRβV29-1 or TCRβV30.

In some embodiments, the second target molecule is selected from thegroup consisting of BCMA, FcRH5, CD19, CD20, CD22, CD30, CD33, CD38,CD47, CD99, CD123, FcRH5, CLEC12, CD179A, SLAMF7, or NY-ESO1, PDL1,CD47, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostatespecific membrane antigen (PSMA), prostate-specific antigen (PSA),carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature lamininreceptor, TAG-72, BING-4, Calcium-activated chloride channel 2,Cyclin-B1, 9D7, Ep-CAM, EphA3, Her2/neu, Telomerase, SAP-1, Survivin,NY-ESO-1/LAGE-1, PRAME, SSX-2, Melan-A/MART-1, Gp100/pmell7, Tyrosinase,TRP-1/-2, MC1R, b-catenin, BRCA1/2, CDK4, CML66, Fibronectin, p53, Ras,TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1,b-catenin, CDK4, CDC27, a actinin-4, TRP1/gp75, TRP2, gp100,Melan-A/MART1, gangliosides, WT1, EphA3, Epidermal growth factorreceptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1,NPM, OA1, OGT, RCC, RUI1, RUI2, SAGE, TRG, TRP1, TSTA, Folate receptoralpha, L1-CAM, CAIX, gpA33, GD3, GM2, VEGFR, Intergrin, a carbohydrates,IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid,collagen, tenascin C and tenascin W.

In some embodiments, the second domain is an NK cell engager, a T cellengager, a B cell engager, a dendritic cell engager, or a macrophagecell engager.

In some embodiments, the second domain is a T cell engager and whereinthe second target molecule is a TCRβV other than the TCRβV to which thefirst domain binds.

In some embodiments, the second target molecule is not a TCRβV.

In some embodiments, the second target molecule is CD19.

In some embodiments, the second target molecule is CD3.

In some embodiments, the second target molecule is CD123.

In some embodiments, the second domain comprises a tumor-targetingdomain and the second target molecule is a cancer antigen.

In some embodiments, the cancer antigen is a hematological cancerantigen, a solid tumor antigen, a metastatic cancer antigen, a softtissue tumor antigen, a cancer antigen of a metastatic lesion or astromal antigen.

In some embodiments, the cancer antigen is: (i) the solid tumor antigen,wherein the solid tumor is pancreatic cancer, breast cancer, colorectalcancer, lung cancer, skin cancer, ovarian cancer, or liver cancer; or(ii) the hematological cancer antigen, wherein the hematological canceris a B-cell malignancy or a T cell malignancy.

In some embodiments, the cancer antigen is the hematological cancerantigen and the B-cell malignancy or the T cell malignancy is Hodgkin'slymphoma, Non-Hodgkin's lymphoma, acute myeloid leukemia (AML), chronicmyeloid leukemia, myelodysplastic syndrome, multiple myeloma, or acutelymphocytic leukemia.

In some embodiments, the cancer antigen is the hematological cancerantigen and the B-cell malignancy is Hodgkin's lymphoma, wherein theNon-Hodgkin's lymphoma is B cell lymphoma, diffuse large B celllymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle celllymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma,lymphoplasmacytic lymphoma, or hairy cell leukemia.

In some embodiments, the second domain comprises a cytokine moleculeselected from the group consisting of interleukin-2 (IL-2),interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15),interleukin-18 (IL-18), interleukin-21 (IL-21), interferon gamma andfunctional fragments or variants thereof.

In some embodiments, binding of the first domain to the TCRβV andbinding of the second molecule to the target molecule promotes the Tcells to kill cancer cells.

In some embodiments, the target cell is a T cell.

In some embodiments, the target cell is a non-cancer cell.

In some embodiments, the method expands T cells in vivo.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1B shows the alignment of the Antibody A source mouse VH and VLframework 1, CDR 1, framework 2, CDR 2, framework 3, CDR3, and framework4 regions with their respective humanized sequences. Kabat CDRs areshown in bold, Chothia CDRs are shown in italics, and combined CDRs areshown in boxes. The framework positions that were back mutated aredouble underlined. FIG. 1A shows VH sequences for murine Antibody A (SEQID NO: 1) and humanized Antibody A-H (SEQ ID NO: 9). FIG. 1B shows VLsequences for murine Antibody A (SEQ ID NO: 2) and humanized AntibodyA-H (SEQ ID NO: 10 and SEQ ID NO: 11).

FIGS. 2A-2B shows the alignment of the Antibody B source mouse VH and VLframework 1, CDR 1, framework 2, CDR 2, framework 3, CDR3, and framework4 regions with their respective humanized sequences. Kabat CDRs areshown in bold, Chothia CDRs are shown in italics, and combined CDRs areshown in boxes. The framework positions that were back mutated aredouble underlined. FIG. 2A shows the VH sequence for murine Antibody B(SEQ ID NO: 15) and humanized VH sequences B-H.1A to B-H.1C (SEQ ID NOs:23-25). FIG. 2B shows the VL sequence for murine Antibody B (SEQ ID NO:16) and humanized VL sequences B-H.1D to B-H.1H (SEQ ID NOs: 26-30).

FIG. 3 depicts the phylogenetic tree of TCRBV gene family andsubfamilies with corresponding antibodies mapped. Subfamily identitiesare as follows: Subfamily A: TCRβ V6; Subfamily B: TCRβ V10; SubfamilyC: TCRβ V12; Subfamily D: TCRβ V5; Subfamily E: TCRβ V7; Subfamily F:TCRβ V11; Subfamily G: TCRβ V14; Subfamily H: TCRβ V16; Subfamily I:TCRβV18; Subfamily J:TCRβ V9; Subfamily K: TCRβ V13; Subfamily L: TCRβ V4;Subfamily M:TCRβ V3; Subfamily N:TCRβ V2; Subfamily O:TCRβ V15;Subfamily P: TCRβ V30; Subfamily Q: TCRβ V19; Subfamily R:TCRβ V27;Subfamily S:TCRβ V28; Subfamily T: TCRβ V24; Subfamily U: TCRβ V20;Subfamily V: TCRβ V25; and Subfamily W:TCRβ V29 subfamily. Subfamilymembers are described in detail herein in the Section titled “TCR beta V(TCRβV)”.

FIGS. 4A-4C show human CD3+ T cells activated by anti-TCR Vβ13.1antibody (A-H.1) for 6-days. Human CD3+ T cells were isolated usingmagnetic-bead separation (negative selection) and activated withimmobilized (plate-coated) anti-TCR Vβ13.1 (A-H.1) or anti-CD3ϵ (OKT3)antibodies at 100 nM for 6 days. FIG. 4A shows two scatter plots (left:activated with OKT3; and right: activated with A-H.1) of expanded Tcells assessed for TCR Vβ13.1 surface expression using anti-TCR Vβ13.1(A-H.1) followed by a secondary fluorochrome-conjugated antibody forflow cytometry analysis. FIG. 4B shows percentage (%) of TCR Vβ13.1positive T cells activated by anti-TCR Vβ13.1 (A-H.1) or anti-CD3e(OKT3) plotted against total T cells (CD3+). FIG. 4C shows relative cellcount acquired by counting the number of events in each T cell subsetgate (CD3 or TCR Vβ13.1) for 20 seconds at a constant rate of 600/min.Data shown as mean value from 3 donors.

FIGS. 5A-5B show cytolytic activity of human CD3+ T cells activated byanti-TCR Vβ13.1 antibody (A-H.1) against transformed cell line RPMI8226. FIG. 5A depicts target cell lysis of human CD3+ T cells activatedwith A-H.1 or OKT3. Human CD3+ T cells were isolated using magnetic-beadseparation (negative selection) and activated with immobilized(plate-coated) A-H.1 or OKT3 at the indicated concentrations for 4 daysprior to co-culture with RPMI 8226 cells at a (E:T) ratio of 5:1 for 2days. Samples were next analyzed for cell lysis of RPMI 8226 cells byFACS staining for CFSE/CD138-labeled, and membrane-impermeable DNA dyes(DRAQ7) using flow cytometry analysis. FIG. 5B shows target cell lysisof human CD3+ T cells activated with A-H.1 or OKT3 incubated withRPMI-8226 at a (E:T) ratio of 5:1 for 6 days followed by cell lysisanalysis of RPMI 8226 cells as described above. Percentage (%) targetcell lysis was determined by normalizing to basal target cell lysis(i.e. without antibody treatment) using the following formula,[(x−basal)/(100%−basal), where x is cell lysis of sample]. Data shown isa representative of n=1 donor.

FIGS. 6A-6B show IFNg production by human PBMCs activated with theindicated antibodies. Human PBMCs were isolated from whole blood fromthe indicated number of donors, followed by solid-phase (plate-coated)stimulation with the indicated antibodies at 100 Nm. Supernatant wascollected on Days 1, 2, 3, 5, or 6. FIG. 6A is a graph comparing theproduction of IFNg in human PBMCs activated with the antibodiesindicated activated with anti-TCR Vβ13.1 antibodies (A-H.1 or A-H.2) oranti-CD3e antibodies (OKT3 or SP34-2) on Day 1, 2, 3, 5, or 6post-activation. FIG. 6B shows IFNg production in human PBMCs activatedwith the antibodies indicated activated with the indicated anti-TCRVβ13.1 antibodies or anti-CD3e antibody (OKT3) on Day 1, 2, 3, 5, or 6post-activation.

FIGS. 7A-7B show IL-2 production by human PBMCs activated with theindicated antibodies. A similar experimental setup as described forFIGS. 6A-6B was used.

FIGS. 8A-8B show IL-6 production by human PBMCs activated with theindicated antibodies. A similar experimental setup as described forFIGS. 6A-6B was used.

FIGS. 9A-9B show TNF-alpha production by human PBMCs activated with theindicated antibodies. A similar experimental setup as described forFIGS. 6A-6B was used.

FIGS. 10A-10B show IL-1beta production by human PBMCs activated with theindicated antibodies. A similar experimental setup as described forFIGS. 6A-6B was used.

FIGS. 11A-11B are graphs showing delayed kinetics of IFNg secretion inhuman PMBCs activated by anti-TCR Vβ13.1 antibody A-H.1 when compared toPBMCs activated by anti-CD3e antibody OKT3. FIG. 11A shows IFNgsecretion data from 4 donors. FIG. 11B shows IFNg secretion data from 4additional donors. Data shown is representative of n=8 donors.

FIG. 12 depicts increased CD8+ TSCM and Temra T cell subsets in humanPBMCs activated by anti-TCR Vβ13.1 antibodies (A-H.1 or A-H.2) comparedto PBMCs activated by anti-CD3e antibodies (OKT3 or SP34-2).

FIGS. 13A-13F show characterization of an anti-TCRVb antibody. FIG. 13Ais a graph depicting proliferation of T cells activated with anti-CD3(OKT3) antibody or anti-TCRVb antibody. FIG. 13B shows selectiveexpansion of CD45RA+ effector memory CD8+ and CD4+ T cells (TEMRA) cellswith anti-TCRVb antibodies. Tn=naïve T cell; Tscm=stem cell memory Tcell; Tcm=central memory T cell; Tem=effector memory T cell;Temra=effector memory CD45RA+ T cell. FIG. 13C is a graph showing IFN-gsecretion by PBMCs stimulated with an anti-TCRVb antibody, or anti-CD3antibodies. FIG. 13D shows target cell lysis by T cells stimulated withan anti-TCRVb antibody, or anti-CD3 antibodies. Cells were stimulatedfor 4 days followed by 2 days incubation with multiple myeloma targetcells for assessment of cell killing. FIG. 13E is a graph showingperforin secretion by T cells stimulated with an anti-TCRVb antibody, oran anti-CD3 antibody. Perforin was analyzed by FACS staining inTCRVB-positive and TCRVB-negative T cells in PBMCs after 5 days ofstimulation with 100 ng/ml plate-bound antibody. FIG. 13F is a graphshowing Granzyme B by T cells stimulated with an anti-TCRVb antibody, oran anti-CD3 antibody. Granzyme B was analyzed by FACS staining inTCRVB-positive and TCRVB-negative T cells in PBMCs after 5 days ofstimulation with 100 ng/ml plate-bound antibody.

FIGS. 14A-14B show production of IL-2 and IL-15 and expansion of humanNK cells by stimulation of PBMCs with anti-TCRVb antibody for 6 days ata dose of 100 nM. FIG. 14A shows secretion of IL-2 or IL-15 in T cellsstimulated with an anti-TCRVb antibody, or anti-CD3 antibodies. FIG. 14Bdepicts flow cytometry dot plots showing NKp46 staining vs CD56 antibodystaining in cells stimulated with an anti-TCRVb antibody or an anti-CD3antibody or a control sample.

FIGS. 15A-15C show secretion of cytokines in PBMCs stimulated with ananti-TCRVb antibody, or anti-CD3 antibodies.

FIGS. 16A-16B show killing of MM cells by dual targeting BCMA-TCRvbantibody molecules. FIG. 16A shows in vitro killing by one of thefollowing dual-targeting antibody molecules: BCMA-TCRVb (Molecule I),BCMA-CD3, or Control-TCRVb; or an isotype control. FIG. 16B shows invivo killing of MM cells by a dual-targeting BCM-TCRVb antibody(Molecule I).

FIG. 17 shows lysis of MM target cells with a dual targeting antibody(Molecule E) which recognized FcRH5 on one arm and TCRVb on the otherarm.

FIGS. 18A-18B demonstrate cytokine production from human PBMCs activatedby anti-TCR Vβ8a antibodies (B-H.1) when compared to those activated byanti-CD3ϵ antibodies (OKT3 or SP34-2). FIG. 18A shows that human PBMCsactivated by anti-TCR Vβ8a antibodies (B-H.1) produce similar or reducedlevels of IFNγ. FIG. 18B shows human PBMCs activated by anti-TCR Vβ8aantibodies (B-H.1) produce higher levels of IL-2 when compared to thoseactivated by anti-CD3ϵ antibodies (OKT3 or SP34-2). Data shown isrepresentative of n=6 donors.

FIGS. 19A-19C demonstrate cytokine production from human PBMCs activatedby anti-TCR Vβ8a antibodies (B-H.1). Human PBMCs activated by anti-TCRVβ8a antibodies (B-H.1) do not significantly produce IL-6 (FIG. 19A),IL1b (FIG. 19B), and less TNFa (FIG. 19C), when compared to PBMCsactivated by anti-CD3ϵ antibodies (OKT3 or SP34-2). Data shown isrepresentative of n=6 donors.

FIGS. 20A-20E demonstrate cytokine production from human PBMCs activatedby anti-TCRβV Antibody D antibody compared to control anti-CD3e antibody(OKT3). FIG. 20A shows that human PBMCs activated by anti-TCRβV AntibodyD antibody produce similar or reduced levels of IFNγ. FIG. 20B showshuman PBMCs activated by anti-TCRβV Antibody D antibody produce higherlevels of IL-2 when compared to those activated by anti-CD3ϵ antibodies(OKT3). Human PBMCs activated by anti-TCRβV Antibody D antibody do notsignificantly produce IL-1beta (FIG. 20C), IL-6, (FIG. 20D), or TNFalpha(FIG. 20E). Data shown is representative of n=4 donors.

FIGS. 21A-21B demonstrate cytokine production from human PBMCs activatedby anti-TCR Vβ5 antibody (Antibody E). FIG. 21A shows that human PBMCsactivated by anti-TCR Vβ5 antibody produce similar or reduced levels ofIFNγ compared to PBMCS activated by anti-CD3ϵ antibodies (OKT3 orSP34-2). FIG. 21B shows human PBMCs activated by the anti-TCR Vβ5 1antibody produce higher levels of IL-2 when compared to those activatedby anti-CD3ϵ antibodies (OKT3 or SP34-2). Data shown is representativeof n=4 donors.

FIGS. 22A-22D demonstrate cytokine production from human PBMCs activatedby an anti-TCR Vβ5 antibody (Antibody E). Human PBMCs activated byanti-TCR Vβ5 antibody do not significantly produce IL-1beta (FIG. 22A),IL-6, (FIG. 22B), TNFalpha (FIG. 22C), or IL-10 (FIG. 22D) as comparedto PBMCs activated by anti-CD3ϵ antibodies (OKT3 or SP34-2). Data shownis representative of n=4 donors.

FIGS. 23A-23F demonstrate cytokine production from human PBMCs activatedby a dual targeting (bispecific molecule) comprising an anti-TCRβVbinding moiety and a BCMA binding moiety. FIG. 23A shows that humanPBMCs activated by the bispecific molecule produce similar or reducedlevels of IFNγ as PBMCS activated by anti-CD3ϵ antibodies (OKT3). FIG.23B shows human PBMCs activated by the bispecific molecule producehigher levels of IL-2 when compared to PBMCs activated by anti-CD3ϵantibodies (OKT3). Human PBMCs activated by the bispecific molecule donot significantly produce IL-1beta (FIG. 23C), IL-6, (FIG. 23D),TNFalpha (FIG. 23E), or IL-10 (FIG. 23F). Data shown is representativeof n=3 donors.

FIGS. 24A-24B show the structure and sequence of eight TCRβV proteinsfrom seven different subfamilies: TCRβV6 subfamily (TCRβV6-5 andTCRβV6-4 are shown), TCRβV28 subfamily, TCRβV19 subfamily, TCRβV9subfamily, TCRβV5 subfamily, TCRβV20 subfamily and TCRβV12 subfamily.FIG. 24A shows the structural alignment of the different TCRβV proteins.The circled area represents the outward facing region comprising theproposed binding site for the anti-TCRβV antibodies disclosed herein.FIG. 24B shows the amino acid sequence alignment of the proteins shownin FIG. 24A (SEQ ID NOS 3449-3456, respectively, in order ofappearance). The various TCRβV proteins (from 7 different TCRβVsubfamilies) have diverse sequences but share a conserved (similar)structure and function.

FIGS. 25A-25J show cytokine or chemokine secretion of PBMCs activatedwith anti-TCRVb antibodies (A-H.1, B-H.1), a bispecific moleculecomprising an anti-TCRVb antibody (Molecule H), control isotype (122) oranti-CD3e antibody (OKT3). Data shown is representative of n=2 donorsand representative of 2 independent experiments.

FIGS. 26A-26H show cytokine or chemokine secretion of PBMCs activatedwith anti-TCRVb antibodies (A-H.1, B-H.1), a bispecific moleculecomprising an anti-TCRVb antibody (Molecule H), control isotype (122) oranti-CD3e antibody (OKT3). Data shown is representative of n=2 donorsand representative of 2 independent experiments.

FIGS. 27A-27L show cytokine or chemokine secretion of PBMCs activatedwith anti-TCRVb antibodies (A-H.1, B-H.1), a bispecific moleculecomprising an anti-TCRVb antibody (Molecule H), control isotype (122) oranti-CD3e antibody (OKT3). Data shown is representative of n=2 donorsand representative of 2 independent experiments.

FIG. 28 is a graph depicting mean tumor volume in NOD/SCID/IL-2Rγnull(NSG) mice engrafted with Raji-luc cells at days 10 to 28. The Stardenotes PBMC implantation. Open triangles denote antibody treatment withthe indicated antibodies.

FIGS. 29A-29B depicting Mean tumor burden (Total Flux) inNOD/SCID/IL-2Rγnull (NSG) mice engrafted with cancer cells and treatedwith the indicated antibody. NSG mice were implanted with PBMCs on Day 1followed by injection with cancer cells on Day 7 (Raji-luc in FIG. 29A;K562-Luc control in FIG. 29B). Antibody treatment with the indicatedantibodies began on Day 16. FIG. 29A shows mean tumor burden at days 16to 37 in NOD/SCID/IL-2Rγnull (NSG) mice engrafted with Raji-luc cells.FIG. 29B shows mean tumor burden (Total Flux) at days 16 to 30 inanimals engrafted with K562-luc cells.

FIG. 30 is a graph depicting Mean tumor burden (Total Flux) mean tumorvolume in NOD/SCID/IL-2Rγnull (NSG) mice engrafted with RPMI-8226 cells.The RPMI-8226 cells were engrafted on Day 1. On Day 11, PBMCs wereimplanted into the mice and antibody treatment began on Day 17.

FIGS. 31A-31B are graphs showing % target cell lysis at differentantibody concentrations. FIG. 31A shows data generated using anti-TCRVβ13.1/anti-CD19 (Molecule F), anti-CD3/anti-CD19, and anti-TCR Vβ13.1(A-H.1). FIG. 31B shows data generated using anti-TCR Vβ13.1/anti-BCMA(Molecule G), anti-CD3/anti-BCMA, and anti-TCR Vβ13.1 (A-H.1).

FIGS. 32A-32F are graphs showing cytokine secretion stimulated byanti-TCR V13/anti-BCMA (Molecule H) or anti-CD3 (OKT3) at Days 1, 2, 3,and 5. Cytokines examined include: IFNγ, IL-2, IL-1β, IL-6, IL-10, andTNFα (FIGS. 32A-32F, respectively).

FIGS. 33A-33F are graphs showing cytokine secretion stimulated byanti-TRBC1 (Antibody F) or anti-CD3 (OKT3) at Days 2 and 5. Cytokinesexamined include: IFNγ, IL-2, IL-1β, IL-6, IL-10, and TNFα (FIGS.33A-33F, respectively).

FIG. 34 is Table 9 showing alignment of TCRBV amino acid sequences (SEQID NOS 3457-3639, respectively, in order of appearance). The alignmentof TCRBV amino acid sequences in Table 9 underscores the diversity ofTCR sequences. In particular, the TRBV sequences from differentsubfamilies are considerably different from each other.

FIGS. 35A and 35B show alignment of affinity matured humanized AntibodyA-H VL sequences (SEQ ID NOS 3377-3389, respectively, in order ofappearance) (FIG. 35A) and alignment of affinity matured humanizedAntibody A-H VH sequences (SEQ ID NOS 3390-3436, respectively, in orderof appearance) (FIG. 35B), respectively.

DETAILED DESCRIPTION OF THE INVENTION

Current bispecific constructs designed to redirect T cells to promotetumor cell lysis for cancer immunotherapy typically utilize antibodyfragments (Fab, scFv, VH, etc.) that are derived from monoclonalantibodies (mAb) directed against the CD3e subunit of the T cellreceptor (TCR). However, there are limitations to this approach whichmay prevent the full realization of the therapeutic potential for suchbispecific constructs. Previous studies have shown that even low“activating” doses of anti-CD3e mAb can cause long-term T celldysfunction and exert immunosuppressive effects. In addition, anti-CD3emAbs have been associated with side effects that result from massive Tcell activation. The large number of activated T cells secretesubstantial amounts of cytokines, the most important of which isInterferon gamma (IFNg). This excess amount of IFNg in turn activatesmacrophages which then overproduce proinflammatory cytokines such asIL-1beta, IL-6, IL-10 and TNF-alpha, causing a “cytokine storm” known asthe cytokine release syndrome (CRS) (Shimabukuro-Vornhagen et al., JImmunother Cancer. 2018 Jun. 15; 6(1):56, herein incorporated byreference in its entirety). Thus, the need exists for developingantibodies that are capable of binding and activating only a subset ofeffector T cells, e.g., to reduce the CRS and/or neurotoxicity (NT).

This invention features molecules targeting the TCRβV chain of TCR andmethods thereof. Without wishing to be bound by theory, such moleculesare capable of binding, activating, and/or expanding only a subset of Tcells, avoiding or reducing CRS and/or NT and minimizing potentialimmunosuppressive effects of anti-CD3 mAbs.

TCR is a disulfide-linked membrane-anchored heterodimeric proteinnormally consisting of the highly variable alpha (α) and beta (β) chainsexpressed as part of a complex with the invariant CD3 chain molecules.TCR on αβ T cells is formed by a heterodimer of one alpha chain and onebeta chain. Each alpha or beta chain consists of a constant domain and ahighly variable domain classified as the Immunoglobulin superfamily(IgSF) fold. The TCRβV chains can be further classified into 30subfamilies (TRBV1-30). Despite their high structural and functionalhomology, the amino acid sequence homology in the TRBV genes is verylow. Only 4 amino acids out of ˜95 are identical while 10 additionalamino acids are conserved among all subfamilies (see an alignment ofTCRBV amino acid sequences in Table 9). Nevertheless, TCRs formedbetween alpha and beta chains of highly diverse sequences show aremarkable structural homology (FIGS. 24A and 24B) and elicit a similarfunction, e.g., activation of T cells.

Disclosed herein is the discovery of a novel class of antibodies, i.e.,anti-TCRBV antibody molecules disclosed herein, which despite having lowsequence similarity (e.g., low sequence identity among the differentantibody molecules that recognize different TCRBV subfamilies),recognize a structurally conserved, yet sequence-wise variable, region,e.g., domain, on the TCRβV protein (as denoted by the circled area inFIG. 24A) and have a similar function (e.g., activation of T cells and asimilar cytokine profile as described herein). Thus, the anti-TCRBVantibody molecules disclosed herein share a structure-functionrelationship.

Without wishing to be bound by theory, it is believed that in someembodiments, the anti-TCRβV antibody molecules disclosed herein bind toan outward facing epitope of a TCRβV protein when it is in a complexwith a TCRalpha protein, e.g., as denoted by the circled area in FIG.24A. In some embodiments, the anti-TCRβV antibody molecules disclosedherein recognize (e.g., bind to), a domain (e.g., an epitope) on theTCRβV protein that is: (1) structurally conserved among different TCRβVsubfamilies; and (2) has minimal sequence identity among the differentTCRβV subfamilies. As shown in Table 9, TCRβV proteins from thedifferent TCRBV subfamilies share minimal sequence similarity. However,as shown in FIG. 24A-B, TCRBV proteins which have minimal sequencesimilarity, share a similar 3D conformation and structure.

In some embodiments, the anti-TCRβV antibody molecules disclosed hereindo not recognize, e.g., bind to, an interface of a TCRβV:TCRalphacomplex.

In some embodiments, the anti-TCRβV antibody molecules disclosed hereindo not recognize, e.g., bind to, a constant region of a TCRβV protein.

In some embodiments, the anti-TCRβV antibody molecules disclosed hereindo not recognize, e.g., bind to, one or more (e.g., all) of acomplementarity determining region (e.g., CDR1, CDR2 and/or CDR3) of aTCRβV protein.

This disclosure provides, inter alia, antibody molecules directed to thevariable chain of the beta subunit of TCR (TCRBV) which bind and, e.g.,activate a subset of T cells. The anti-TCRβV antibody moleculesdisclosed herein result in lesser or no production of cytokinesassociated with CRS, e.g., IL-6, IL-1beta, IL-10 and TNF alpha; andenhanced and/or delayed production of IL-2 and IFNg. In someembodiments, the anti-TCRβV antibodies disclosed herein have a cytokineprofile, e.g., as described herein, which differs from a cytokineprofile of a T cell engager that binds to a receptor or molecule otherthan a TCRβV region (“a non-TCRβV-binding T cell engager”). In someembodiments, the anti-TCRBV antibodies disclosed herein result inexpansion of TCRβV+ T cells, e.g., a subset of memory effector T cellsknown as T_(EMRA). Without wishing to be bound by theory, it is believedthat in some embodiments, T_(EMRA) cells can promote tumor cell lysisbut not CRS. Accordingly, provided herein are methods of making saidanti-TCRBV antibody molecules and uses thereof. Also disclosed hereinare multispecific molecules, e.g., bispecific molecules comprising saidanti-TCRβV antibody molecules. In some embodiments, compositionscomprising anti-TCRβV antibody molecules of the present disclosure, canbe used, e.g., to: (1) activate and redirect T cells to promote tumorcell lysis for cancer immunotherapy; and/or (2) expand TCRβV+ T cells.In some embodiments, compositions comprising anti-TCRβV antibodymolecules as disclosed herein limit the harmful side-effects of CRSand/or NT, e.g., CRS and/or NT associated with anti-CD3e targeting.

In some embodiments, the anti-TCRβV antibody molecule does not bind toTCRβ V12, or binds to TCRβ V12 with an affinity and/or bindingspecificity that is less than (e.g., less than about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/orbinding specificity of the 16G8 murine antibody or a humanized versionthereof as described in U.S. Pat. No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to TCRβ V12with an affinity and/or binding specificity that is greater than (e.g.,greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about2-, 5-, or 10-fold) the affinity and/or binding specificity of the 16G8murine antibody or a humanized version thereof as described in U.S. Pat.No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβVregion other than TCRβ V12 (e.g., TCRβV region as described herein,e.g., TCRβ V6 subfamily (e.g., TCRβ V6-5*01) with an affinity and/orbinding specificity that is greater than (e.g., greater than about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) theaffinity and/or binding specificity of the 16G8 murine antibody or ahumanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule does not comprisethe CDRs of the Antibody B murine antibody.

In some embodiments, the anti-TCRβV antibody molecule does not bind toTCRβ V5-5*01 or TCRβ V5-1*01, or binds to TCRβ V5-5*01 or TCRβ V5-1*01with an affinity and/or binding specificity that is less than (e.g.,less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-,5-, or 10-fold) the affinity and/or binding specificity of the TM23murine antibody or a humanized version thereof as described in U.S. Pat.No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to TCRβV5-5*01 or TCRβ V5-1*01 with an affinity and/or binding specificity thatis greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/or bindingspecificity of the TM23 murine antibody or a humanized version thereofas described in U.S. Pat. No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβVregion other than TCRβ V5-5*01 or TCRβ V5-1*01 (e.g., TCRβV region asdescribed herein, e.g., TCRβ V6 subfamily (e.g., TCRβ V6-5*01) with anaffinity and/or binding specificity that is greater than (e.g., greaterthan about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-,or 10-fold) the affinity and/or binding specificity of the TM23 murineantibody or a humanized version thereof as described in U.S. Pat. No.5,861,155.

In some embodiments, the anti-TCRβV antibody molecule does not comprisethe CDRs of the TM23 murine antibody.

Accordingly, provided herein are, inter alia, anti-TCRβV antibodymolecules, multispecific or multifunctional molecules (e.g.,multispecific or multifunctional antibody molecules) that compriseanti-TCRβV antibody molecules, nucleic acids encoding the same, methodsof producing the aforesaid molecules, pharmaceutical compositionscomprising aforesaid molecules, and methods of treating a disease ordisorder, e.g., cancer, using the aforesaid molecules. The antibodymolecules and pharmaceutical compositions disclosed herein can be used(alone or in combination with other agents or therapeutic modalities) totreat, prevent and/or diagnose disorders and conditions, e.g., cancer,e.g., as described herein.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains.

The term “a” and “an” refers to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations of±20% or in some instances ±10%, or in some instances ±5%, or in someinstances ±1%, or in some instances ±0.1% from the specified value, assuch variations are appropriate to perform the disclosed methods.

The term “acquire” or “acquiring” as the terms are used herein, refer toobtaining possession of a physical entity (e.g., a sample, apolypeptide, a nucleic acid, or a sequence), or a value, e.g., anumerical value, by “directly acquiring” or “indirectly acquiring” thephysical entity or value. “Directly acquiring” means performing aprocess (e.g., performing a synthetic or analytical method) to obtainthe physical entity or value. “Indirectly acquiring” refers to receivingthe physical entity or value from another party or source (e.g., a thirdparty laboratory that directly acquired the physical entity or value).Directly acquiring a physical entity includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial. Directly acquiring a value includes performing a process thatincludes a physical change in a sample or another substance, e.g.,performing an analytical process which includes a physical change in asubstance, e.g., a sample.

As used herein, the term “T cell receptor beta variable chain” or“TCRβV,” refers to an extracellular region of the T cell receptor betachain which comprises the antigen recognition domain of the T cellreceptor. The term TCRβV includes isoforms, mammalian, e.g., humanTCRβV, species homologs of human and analogs comprising at least onecommon epitope with TCRβV. Human TCRβV comprises a gene familycomprising subfamilies including, but not limited to: a TCRβ V6subfamily, a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5subfamily, a TCRβ V7 subfamily, a TCRβ V11 subfamily, a TCRβ V14subfamily, a TCRβ V16 subfamily, a TCRβ V18 subfamily, a TCRβ V9subfamily, a TCRβ V13 subfamily, a TCRβ V4 subfamily, a TCRβ V3subfamily, a TCRβ V2 subfamily, a TCRβ V15 subfamily, a TCRβ V30subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβ V28subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25subfamily, a TCRβ V29 subfamily, a TCRβ V1 subfamily, a TCRβ V17subfamily, a TCRβ V21 subfamily, a TCRβ V23 subfamily, or a TCRβ V26subfamily, as well as family members of said subfamilies, and variantsthereof (e.g., a structural or functional variant thereof). In someembodiments, the TCR3 V6 subfamily comprises: TCRβ V6-4*01, TCRβV6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβV6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCR3 V6-1*01. In someembodiments, TCRβV comprises TCRβ V6-5*01, or a variant thereof, e.g., avariant having 85%, 90%, 95%, 99% or more identity thenaturally-occurring sequence. TCRβ V6-5*01 is also known as TRBV65;TCRBV6S5; TCRBV13S1, or TCRβ V13.1. The amino acid sequence of TCRβV6-5*01, e.g., human TCRβ V6-5*01, is known in that art, e.g., asprovided by IMGT ID L36092. In some embodiments, TCRβ V6-5*01 is encodedby the nucleic acid sequence of SEQ ID NO: 43, or a sequence having 85%,90%, 95%, 99% or more identity thereof. In some embodiments, TCRβV6-5*01 comprises the amino acid sequence of SEQ ID NO: 44, or asequence having 85%, 90%, 95%, 99% or more identity thereof.

The term “human-like antibody molecule” as used herein refers to ahumanized antibody molecule, human antibody molecule or an antibodymolecule having at least 95% identity with a non-murine germlineframework region, e.g., FR1, FR2, FR3 and/or FR4. In some embodiments,the human-like antibody molecule comprises a framework region having atleast 95% identity to a human germline framework region, e.g., a FR1,FR2, FR3 and/or FR4 of a human germline framework region. In someembodiments, the human-like antibody molecule is a recombinant antibody.In some embodiments, the human-like antibody molecule is a humanizedantibody molecule. In some embodiments, the human-like antibody moleculeis human antibody molecule. In some embodiments, the human-like antibodymolecule is a phage display or a yeast display antibody molecule. Insome embodiments, the human-like antibody molecule is a chimericantibody molecule. In some embodiments, the human-like antibody moleculeis a CDR grafted antibody molecule.

The term “cytokine profile” as used herein, refers to the level and/oractivity of on one or more cytokines or chemokines, e.g., as describedherein. In some embodiments, a cytokine profile comprises the leveland/or activity of a naturally occurring cytokine, a fragment or avariant thereof. In an embodiment, a cytokine profile comprises thelevel and/or activity of one or more cytokines and/or one or morechemokines (e.g., as described herein). In some embodiments, a cytokineprofile comprises the level and/or activity of a naturally occurringcytokine, a fragment or a variant thereof. In some embodiments, acytokine profile comprises the level and/or activity of a naturallyoccurring chemokine, a fragment or a variant thereof. In an embodiment,a cytokine profile comprises the level and/or activity of one or moreof: IL-2 (e.g., full length, a variant, or a fragment thereof); IL-1beta(e.g., full length, a variant, or a fragment thereof); IL-6 (e.g., fulllength, a variant, or a fragment thereof); TNFα (e.g., full length, avariant, or a fragment thereof); IFNg (e.g., full length, a variant, ora fragment thereof) IL-10 (e.g., full length, a variant, or a fragmentthereof); IL-4 (e.g., full length, a variant, or a fragment thereof);TNF alpha (e.g., full length, a variant, or a fragment thereof);IL-12p70 (e.g., full length, a variant, or a fragment thereof); IL-13(e.g., full length, a variant, or a fragment thereof); IL-8 (e.g., fulllength, a variant, or a fragment thereof); Eotaxin (e.g., full length, avariant, or a fragment thereof); Eotaxin-3 (e.g., full length, avariant, or a fragment thereof); IL-8 (HA) (e.g., full length, avariant, or a fragment thereof); IP-10 (e.g., full length, a variant, ora fragment thereof); MCP-1 (e.g., full length, a variant, or a fragmentthereof); MCP-4 (e.g., full length, a variant, or a fragment thereof);MDC (e.g., full length, a variant, or a fragment thereof); MIP-1a (e.g.,full length, a variant, or a fragment thereof); MIP-1b (e.g., fulllength, a variant, or a fragment thereof); TARC (e.g., full length, avariant, or a fragment thereof); GM-CSF (e.g., full length, a variant,or a fragment thereof); IL-12 23p40 (e.g., full length, a variant, or afragment thereof); IL-15 (e.g., full length, a variant, or a fragmentthereof); IL-16 (e.g., full length, a variant, or a fragment thereof);IL-17a (e.g., full length, a variant, or a fragment thereof); IL-1a(e.g., full length, a variant, or a fragment thereof); IL-5 (e.g., fulllength, a variant, or a fragment thereof); IL-7 (e.g., full length, avariant, or a fragment thereof); TNF-beta (e.g., full length, a variant,or a fragment thereof); or VEGF (e.g., full length, a variant, or afragment thereof). In some embodiments, a cytokine profile includessecretion of one or more cytokines or chemokines.

In an embodiment, a cytokine in a cytokine profile can be modulated,e.g., increased or decreased, by an anti-TCRBV antibody moleculedescribed herein. In one embodiment, the cytokine profile includescytokines associated with a cytokine storm or cytokine release syndrome(CRS), e.g., IL-6, IL-1beta, TNFalpha and IL-10.

The term “variant” refers to a polypeptide that has a substantiallyidentical amino acid sequence to the naturally-occurring sequence, orare encoded by a substantially identical nucleotide sequence. In someembodiments, the variant is a functional variant. In some embodiments, aTCRβV variant can bind to TCRα and form a TCR α:β complex.

The term “functional variant” refers to a polypeptide that has asubstantially identical amino acid sequence to the naturally-occurringsequence, or are encoded by a substantially identical nucleotidesequence, and are capable of having one or more activities of thenaturally-occurring sequence.

As used herein, a “multifunctional” or a “multispecific” molecule refersto molecule, e.g., a polypeptide, that has two or more functionalities,e.g., two or more binding specificities. In some embodiments, thefunctionalities can include one or more immune cell engagers, one ormore tumor binding molecules, one or more cytokine molecules, one ormore stromal modifiers, and other moieties described herein. In someembodiments, the multispecific molecule is a multispecific antibodymolecule, e.g., a bispecific antibody molecule. In some embodiments, themultispecific molecule includes an anti-TCRVb antibody molecule asdescribed herein.

In some embodiments, the multifunctional molecule includes an immunecell engager. “An immune cell engager” refers to one or more bindingspecificities that bind and/or activate an immune cell, e.g., a cellinvolved in an immune response. In embodiments, the immune cell ischosen from a T cell, an NK cell, a B cell, a dendritic cell, and/or themacrophage cell. The immune cell engager can be an antibody molecule, areceptor molecule (e.g., a full length receptor, receptor fragment, orfusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule(e.g., a full length ligand, ligand fragment, or fusion thereof (e.g., aligand-Fc fusion)) that binds to the immune cell antigen (e.g., the Tcell, the NK cell antigen, the B cell antigen, the dendritic cellantigen, and/or the macrophage cell antigen). In embodiments, the immunecell engager specifically binds to the target immune cell, e.g., bindspreferentially to the target immune cell. For example, when the immunecell engager is an antibody molecule, it binds to an immune cell antigen(e.g., a T cell antigen, an NK cell antigen, a B cell antigen, adendritic cell antigen, and/or a macrophage cell antigen) with adissociation constant of less than about 10 nM.

In some embodiments, the multifunctional molecule includes a cytokinemolecule. As used herein, a “cytokine molecule” refers to full length, afragment or a variant of a cytokine; a cytokine further comprising areceptor domain, e.g., a cytokine receptor dimerizing domain; or anagonist of a cytokine receptor, e.g., an antibody molecule (e.g., anagonistic antibody) to a cytokine receptor, that elicits at least oneactivity of a naturally-occurring cytokine. In some embodiments thecytokine molecule is chosen from interleukin-2 (IL-2), interleukin-7(IL-7), interleukin-12 (IL-12), interleukin-10 (IL-10), interleukin-15(IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or interferongamma, or a fragment or variant thereof, or a combination of any of theaforesaid cytokines. The cytokine molecule can be a monomer or a dimer.In embodiments, the cytokine molecule can further include a cytokinereceptor dimerizing domain. In other embodiments, the cytokine moleculeis an agonist of a cytokine receptor, e.g., an antibody molecule (e.g.,an agonistic antibody) to a cytokine receptor chosen from an IL-15Ra orIL-21R.

As used herein, the term “molecule” as used in, e.g., antibody molecule,cytokine molecule, receptor molecule, includes full-length,naturally-occurring molecules, as well as variants, e.g., functionalvariants (e.g., truncations, fragments, mutated (e.g., substantiallysimilar sequences) or derivatized form thereof), so long as at least onefunction and/or activity of the unmodified (e.g., naturally-occurring)molecule remains.

In some embodiments, the multifunctional molecule includes a stromalmodifying moiety. A “stromal modifying moiety,” as used herein refers toan agent, e.g., a protein (e.g., an enzyme), that is capable ofaltering, e.g., degrading a component of, the stroma. In embodiments,the component of the stroma is chosen from, e.g., an ECM component,e.g., a glycosaminoglycan, e.g., hyaluronan (also known as hyaluronicacid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparinsulfate, heparin, entactin, tenascin, aggrecan and keratin sulfate; oran extracellular protein, e.g., collagen, laminin, elastin, fibrinogen,fibronectin, and vitronectin.

Certain terms are defined below.

As used herein, the articles “a” and “an” refer to one or more than one,e.g., to at least one, of the grammatical object of the article. The useof the words “a” or “an” when used in conjunction with the term“comprising” herein may mean “one,” but it is also consistent with themeaning of “one or more,” “at least one,” and “one or more than one.”

As used herein, “about” and “approximately” generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Exemplary degrees of error are within 20 percent(%), typically, within 10%, and more typically, within 5% of a givenrange of values.

“Antibody molecule” as used herein refers to a protein, e.g., animmunoglobulin chain or fragment thereof, comprising at least oneimmunoglobulin variable domain structure and/or sequence. An antibodymolecule encompasses antibodies (e.g., full-length antibodies) andantibody fragments. In an embodiment, an antibody molecule comprises anantigen binding or functional fragment of a full length antibody, or afull length immunoglobulin chain. For example, a full-length antibody isan immunoglobulin (Ig) molecule (e.g., an IgG antibody) that isnaturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes). In embodiments, an antibody molecule refersto an immunologically active, antigen-binding portion of animmunoglobulin molecule, such as an antibody fragment. An antibodyfragment, e.g., functional fragment, is a portion of an antibody, e.g.,Fab, Fab ‘, F(ab’)₂, F(ab)₂, variable fragment (Fv), domain antibody(dAb), or single chain variable fragment (scFv). A functional antibodyfragment binds to the same antigen as that recognized by the intact(e.g., full-length) antibody. The terms “antibody fragment” or“functional fragment” also include isolated fragments consisting of thevariable regions, such as the “Fv” fragments consisting of the variableregions of the heavy and light chains or recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (“scFv proteins”). In some embodiments, anantibody fragment does not include portions of antibodies withoutantigen binding activity, such as Fc fragments or single amino acidresidues. Exemplary antibody molecules include full length antibodiesand antibody fragments, e.g., dAb (domain antibody), single chain, Fab,Fab′, and F(ab′) 2 fragments, and single chain variable fragments(scFvs). In some embodiments, the antibody molecule is an antibodymimetic. In some embodiments, the antibody molecule is, or comprises, anantibody-like framework or scaffold, such as, fibronectins, ankyrinrepeats (e.g., designed ankyrin repeat proteins (DARPins)), avimers,affibody affinity ligands, anticalins, or affilin molecules.

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence which can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may or may not include one, two, or more N- orC-terminal amino acids, or may include other alterations that arecompatible with formation of the protein structure.

In embodiments, an antibody molecule is monospecific, e.g., it comprisesbinding specificity for a single epitope. In some embodiments, anantibody molecule is multispecific, e.g., it comprises a plurality ofimmunoglobulin variable domain sequences, where a first immunoglobulinvariable domain sequence has binding specificity for a first epitope anda second immunoglobulin variable domain sequence has binding specificityfor a second epitope. In some embodiments, an antibody molecule is abispecific antibody molecule. “Bispecific antibody molecule” as usedherein refers to an antibody molecule that has specificity for more thanone (e.g., two, three, four, or more) epitope and/or antigen.

“Antigen” (Ag) as used herein refers to a molecule that can provoke animmune response, e.g., involving activation of certain immune cellsand/or antibody generation. Any macromolecule, including almost allproteins or peptides, can be an antigen. Antigens can also be derivedfrom genomic recombinant or DNA. For example, any DNA comprising anucleotide sequence or a partial nucleotide sequence that encodes aprotein capable of eliciting an immune response encodes an “antigen.” Inembodiments, an antigen does not need to be encoded solely by a fulllength nucleotide sequence of a gene, nor does an antigen need to beencoded by a gene at all. In embodiments, an antigen can be synthesizedor can be derived from a biological sample, e.g., a tissue sample, atumor sample, a cell, or a fluid with other biological components. Asused, herein a “tumor antigen” or interchangeably, a “cancer antigen”includes any molecule present on, or associated with, a cancer, e.g., acancer cell or a tumor microenvironment that can provoke an immuneresponse. As used, herein an “immune cell antigen” includes any moleculepresent on, or associated with, an immune cell that can provoke animmune response.

The “antigen-binding site,” or “binding portion” of an antibody moleculerefers to the part of an antibody molecule, e.g., an immunoglobulin (Ig)molecule, that participates in antigen binding. In embodiments, theantigen binding site is formed by amino acid residues of the variable(V) regions of the heavy (H) and light (L) chains. Three highlydivergent stretches within the variable regions of the heavy and lightchains, referred to as hypervariable regions, are disposed between moreconserved flanking stretches called “framework regions,” (FRs). FRs areamino acid sequences that are naturally found between, and adjacent to,hypervariable regions in immunoglobulins. In embodiments, in an antibodymolecule, the three hypervariable regions of a light chain and the threehypervariable regions of a heavy chain are disposed relative to eachother in three dimensional space to form an antigen-binding surface,which is complementary to the three-dimensional surface of a boundantigen. The three hypervariable regions of each of the heavy and lightchains are referred to as “complementarity-determining regions,” or“CDRs.” The framework region and CDRs have been defined and described,e.g., in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, and Chothia, C. et al.(1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variableheavy chain and variable light chain) is typically made up of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in theamino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

“Cancer” as used herein can encompass all types of oncogenic processesand/or cancerous growths. In embodiments, cancer includes primary tumorsas well as metastatic tissues or malignantly transformed cells, tissues,or organs. In embodiments, cancer encompasses all histopathologies andstages, e.g., stages of invasiveness/severity, of a cancer. Inembodiments, cancer includes relapsed and/or resistant cancer. The terms“cancer” and “tumor” can be used interchangeably. For example, bothterms encompass solid and liquid tumors. As used herein, the term“cancer” or “tumor” includes premalignant, as well as malignant cancersand tumors.

As used herein, an “immune cell” refers to any of various cells thatfunction in the immune system, e.g., to protect against agents ofinfection and foreign matter. In embodiments, this term includesleukocytes, e.g., neutrophils, eosinophils, basophils, lymphocytes, andmonocytes. Innate leukocytes include phagocytes (e.g., macrophages,neutrophils, and dendritic cells), mast cells, eosinophils, basophils,and natural killer cells. Innate leukocytes identify and eliminatepathogens, either by attacking larger pathogens through contact or byengulfing and then killing microorganisms, and are mediators in theactivation of an adaptive immune response. The cells of the adaptiveimmune system are special types of leukocytes, called lymphocytes. Bcells and T cells are important types of lymphocytes and are derivedfrom hematopoietic stem cells in the bone marrow. B cells are involvedin the humoral immune response, whereas T cells are involved incell-mediated immune response. The term “immune cell” includes immuneeffector cells.

“Immune effector cell,” as that term is used herein, refers to a cellthat is involved in an immune response, e.g., in the promotion of animmune effector response. Examples of immune effector cells include, butare not limited to, T cells, e.g., alpha/beta T cells and gamma/delta Tcells, B cells, natural killer (NK) cells, natural killer T (NK T)cells, and mast cells.

The term “effector function” or “effector response” refers to aspecialized function of a cell. Effector function of a T cell, forexample, may be cytolytic activity or helper activity including thesecretion of cytokines.

The compositions and methods of the present invention encompasspolypeptides and nucleic acids having the sequences specified, orsequences substantially identical or similar thereto, e.g., sequences atleast 80%, 85%, 90%, 95% identical or higher to the sequence specified.In the context of an amino acid sequence, the term “substantiallyidentical” is used herein to refer to a first amino acid that contains asufficient or minimum number of amino acid residues that are i)identical to, or ii) conservative substitutions of aligned amino acidresidues in a second amino acid sequence such that the first and secondamino acid sequences can have a common structural domain and/or commonfunctional activity. For example, amino acid sequences that contain acommon structural domain having at least about 80%, 85%, 90%. 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence,e.g., a sequence provided herein.

In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a referencesequence, e.g., a sequence provided herein.

The term “variant” refers to a polypeptide that has a substantiallyidentical amino acid sequence to a reference amino acid sequence, or isencoded by a substantially identical nucleotide sequence. In someembodiments, the variant is a functional variant.

The term “functional variant” refers to a polypeptide that has asubstantially identical amino acid sequence to a reference amino acidsequence, or is encoded by a substantially identical nucleotidesequence, and is capable of having one or more activities of thereference amino acid sequence.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, 60%, and even more preferably at least 70%,80%, 90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”).

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package (available atwww.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and agap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3,4, 5, or 6. In yet another preferred embodiment, the percent identitybetween two nucleotide sequences is determined using the GAP program inthe GCG software package (available at www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set ofparameters (and the one that should be used unless otherwise specified)are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extendpenalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller ((1989)CABIOS, 4:11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and) (BLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecule of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g.,)(BLAST and NBLAST) can be used.

It is understood that the molecules of the present invention may haveadditional conservative or non-essential amino acid substitutions, whichdo not have a substantial effect on their functions.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and allstereoisomers of any of any of the foregoing. As used herein the term“amino acid” includes both the D- or L-optical isomers andpeptidomimetics.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

The terms “polypeptide”, “peptide” and “protein” (if single chain) areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. The polypeptide can be isolatedfrom natural sources, can be a produced by recombinant techniques from aeukaryotic or prokaryotic host, or can be a product of syntheticprocedures.

The terms “nucleic acid,” “nucleic acid sequence,” “nucleotidesequence,” or “polynucleotide sequence,” and “polynucleotide” are usedinterchangeably. They refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. The polynucleotide may be either single-stranded ordouble-stranded, and if single-stranded may be the coding strand ornon-coding (antisense) strand. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Thesequence of nucleotides may be interrupted by non-nucleotide components.A polynucleotide may be further modified after polymerization, such asby conjugation with a labeling component. The nucleic acid may be arecombinant polynucleotide, or a polynucleotide of genomic, cDNA,semisynthetic, or synthetic origin which either does not occur in natureor is linked to another polynucleotide in a non-natural arrangement.

The term “isolated,” as used herein, refers to material that is removedfrom its original or native environment (e.g., the natural environmentif it is naturally occurring). For example, a naturally-occurringpolynucleotide or polypeptide present in a living animal is notisolated, but the same polynucleotide or polypeptide, separated by humanintervention from some or all of the co-existing materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of the environment in which it is found in nature.

Various aspects of the invention are described in further detail below.Additional definitions are set out throughout the specification.

Human T Cell Receptor (TCR) Complex

T cell receptors (TCR) can be found on the surface of T cells. TCRsrecognize antigens, e.g., peptides, presented on, e.g., bound to, majorhistocompatibility complex (MHC) molecules on the surface of cells,e.g., antigen-presenting cells. TCRs are heterodimeric molecules and cancomprise an alpha chain, a beta chain, a gamma chain or a delta chain.TCRs comprising an alpha chain and a beta chain are also referred to asTCRαβ. The TCR beta chain consists of the following regions (also knownas segments): variable (V), diversity (D), joining (J) and constant (C)(see Mayer G. and Nyland J. (2010) Chapter 10: Major HistocompatibilityComplex and T-cell Receptors-Role in Immune Responses. In: Microbiologyand Immunology on-line, University of South Carolina School ofMedicine). The TCR alpha chain consists of V, J and C regions. Therearrangement of the T-cell receptor (TCR) through somatic recombinationof V (variable), D (diversity), J (joining), and C (constant) regions isa defining event in the development and maturation of a T cell. TCR generearrangement takes place in the thymus.

TCRs can comprise a receptor complex, known as the TCR complex, whichcomprises a TCR heterodimer comprising of an alpha chain and a betachain, and dimeric signaling molecules, e.g., CD3 co-receptors, e.g.,CD3δ/ε, and/or CD3γ/ε.

TCR beta V (TCRβV)

Diversity in the immune system enables protection against a huge arrayof pathogens. Since the germline genome is limited in size, diversity isachieved not only by the process of V(D)J recombination but also byjunctional (junctions between V-D and D-J segments) deletion ofnucleotides and addition of pseudo-random, non-templated nucleotides.The TCR beta gene undergoes gene arrangement to generate diversity.

The TCR V beta repertoire varies between individuals and populationsbecause of, e.g., 7 frequently occurring inactivating polymorphisms infunctional gene segments and a large insertion/deletion-relatedpolymorphism encompassing 2 V beta gene segments.

This disclosure provides, inter alia, antibody molecules and fragmentsthereof, that bind, e.g., specifically bind, to a human TCR beta V chain(TCRβV), e.g., a TCRβV gene family (also referred to as a group), e.g.,a TCRβV subfamily (also referred to as a subgroup), e.g., as describedherein. TCR beta V families and subfamilies are known in the art, e.g.,as described in Yassai et al., (2009) Immunogenetics 61(7) pp:493-502;Wei S. and Concannon P. (1994) Human Immunology 41(3) pp: 201-206. Theantibodies described herein can be recombinant antibodies, e.g.,recombinant non-murine antibodies, e.g., recombinant human or humanizedantibodies.

In an aspect, the disclosure provides an anti-TCRβV antibody moleculethat binds to human TCRβV, e.g., a TCRβV family, e.g., gene family or avariant thereof. In some embodiments a TCRBV gene family comprises oneor more subfamilies, e.g., as described herein, e.g., in FIG. 3, Table8A or Table 8B. In some embodiments, the TCRβV gene family comprises: aTCRβ V6 subfamily, a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5subfamily, a TCRβ V7 subfamily, a TCRβ V11 subfamily, a TCRβ V14subfamily, a TCRβ V16 subfamily, a TCRβ V18 subfamily, a TCRβ V9subfamily, a TCRβ V13 subfamily, a TCRβ V4 subfamily, a TCRβ V3subfamily, a TCRβ V2 subfamily, a TCRβ V15 subfamily, a TCRβ V30subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβ V28subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25subfamily, a TCRβ V29 subfamily, a TCRβ V1 subfamily, a TCRβ V17subfamily, a TCRβ V21 subfamily, a TCRβ V23 subfamily, or a TCRβ V26subfamily.

In some embodiments, TCRβ V6 subfamily is also known as TCRβ V13.1. Insome embodiments, the TCRβ V6 subfamily comprises: TCRβ V6-4*01, TCRβV6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβV6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01, or a variantthereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4*01, or avariant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4*02, ora variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-9*01,or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβV6-8*01, or a variant thereof. In some embodiments, TCRβ V6 comprisesTCRβ V6-5*01, or a variant thereof. In some embodiments, TCRβ V6comprises TCRβ V6-6*02, or a variant thereof. In some embodiments, TCRβV6 comprises TCRβ V6-6*01, or a variant thereof. In some embodiments,TCRβ V6 comprises TCRβ V6-2*01, or a variant thereof. In someembodiments, TCRβ V6 comprises TCRβ V6-3*01, or a variant thereof. Insome embodiments, TCRβ V6 comprises TCRβ V6-1*01, or a variant thereof.

In some embodiments, TCRβ V6 comprises TCRβ V6-5*01, or a variantthereof. In some embodiments, TCRβ V6, e.g., TCRβ V6-5*01, isrecognized, e.g., bound, by SEQ ID NO: 1 and/or SEQ ID NO: 2. In someembodiments, TCRβ V6, e.g., TCRβ V6-5*01, is recognized, e.g., bound, bySEQ ID NO: 9 and/or SEQ ID NO: 10. In some embodiments, TCRβ V6 isrecognized, e.g., bound, by SEQ ID NO: 9 and/or SEQ ID NO: 11.

In some embodiments, TCRβ V10 subfamily is also known as TCRβ V12. Insome embodiments, the TCRβ V10 subfamily comprises: TCRβ V10-1*01, TCRβV10-1*02, TCRβ V10-3*01 or TCRβ V10-2*01, or a variant thereof.

In some embodiments, TCRβ V12 subfamily is also known as TCRβ V8.1. Insome embodiments, the TCRβ V12 subfamily comprises: TCRβ V12-4*01, TCRβV12-3*01, or TCRβ V12-5*01, or a variant thereof. In some embodiments,TCRβ V12 is recognized, e.g., bound, by SEQ ID NO: 15 and/or SEQ ID NO:16. In some embodiments, TCRβ V12 is recognized, e.g., bound, by any oneof SEQ ID NOs 23-25, and/or any one of SEQ ID NO: 26-30:

In some embodiments, the TCRβ V5 subfamily is chosen from: TCRβ V5-5*01,TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-8*01, TCRβ V5-1*01, or a variantthereof.

In some embodiments, the TCRβ V7 subfamily comprises TCRβ V7-7*01, TCRβV7-6*01, TCRβ V7-8*02, TCRβ V7-4*01, TCRβ V7-2*02, TCRβ V7-2*03, TCRβV7-2*01, TCRβ V7-3*01, TCRβ V7-9*03, or TCRβ V7-9*01, or a variantthereof.

In some embodiments, the TCRβ V11 subfamily comprises: TCRβ V11-1*01,TCRβ V11-2*01 or TCRβ V11-3*01, or a variant thereof.

In some embodiments, the TCRβ V14 subfamily comprises TCRβ V14*01, or avariant thereof.

In some embodiments, the TCRβ V16 subfamily comprises TCRβ V16*01, or avariant thereof.

In some embodiments, the TCRβ V18 subfamily comprises TCRβ V18*01, or avariant thereof.

In some embodiments, the TCRβ V9 subfamily comprises TCRβ V9*01 or TCRβV9*02, or a variant thereof.

In some embodiments, the TCRβ V13 subfamily comprises TCRβ V13*01, or avariant thereof.

In some embodiments, the TCRβ V4 subfamily comprises TCRβ V4-2*01, TCRβV4-3*01, or TCRβ V4-1*01, or a variant thereof.

In some embodiments, the TCRβ V3 subfamily comprises TCRβ V3-1*01, or avariant thereof.

In some embodiments, the TCRβ V2 subfamily comprises TCRβ V2*01, or avariant thereof.

In some embodiments, the TCRβ V15 subfamily comprises TCRβ V15*01, or avariant thereof.

In some embodiments, the TCRβ V30 subfamily comprises TCRβ V30*01, orTCRβ V30*02, or a variant thereof.

In some embodiments, the TCRβ V19 subfamily comprises TCRβ V19*01, orTCRβ V19*02, or a variant thereof.

In some embodiments, the TCRβ V27 subfamily comprises TCRβ V27*01, or avariant thereof.

In some embodiments, the TCRβ V28 subfamily comprises TCRβ V28*01, or avariant thereof.

In some embodiments, the TCRβ V24 subfamily comprises TCRβ V24-1*01, ora variant thereof.

In some embodiments, the TCRβ V20 subfamily comprises TCRβ V20-1*01, orTCRβ V20-1*02, or a variant thereof.

In some embodiments, the TCRβ V25 subfamily comprises TCRβ V25-1*01, ora variant thereof.

In some embodiments, the TCRβ V29 subfamily comprises TCRβ V29-1*01, ora variant thereof.

TABLE 8A List of TCRβV subfamilies and subfamily members Reference inFIG. 3 Subfamily Subfamily members A TCRβ V6 TCRβ V6-4*01, TCRβ V6-4*02,TCRβ Also referred to V6-9*01, TCRβ V6-8*01, TCRβ as: TCR VB 13.1V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβV6-1*01. B TCRβ V10 TCRβ V10-1*01, TCRβ V10-1*02, TCRβ Also referred toV10-3*01 or TCRβ V10-2*01 as: TCRβ V12 C TCRβ V12 TCRβ V12-4*01, TCRβV12-3*01, or Also referred to TCRβ V12-5*01 as: TCRβ V8.1 D TCRβ V5 TCRβV5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-8*01, TCRβ V5-1*01 E TCRβV7 TCRβ V7-7*01, TCRβ V7-6*01, TCRβ V7 -8*02, TCRβ V7 -4*01, TCRβV7-2*02, TCRβ V7-2*03, TCRβ V7-2*01, TCRβ V7-3*01, TCRβ V7-9*03, or TCRβV7-9*01 F TCRβ V11 TCRβ V11-1*01, TCRβ V11-2*01 or TCRβ V11-3*01 G TCRβV14 TCRβ V14*01 H TCRβ V16 TCRβ V16*01 I TCRβ V18 TCRβ V18*01 J TCRβ V9TCRβ V9*01 or TCRβ V9*02 K TCRβ V13 TCRβ V13*01 L TCRβ V4 TCRβ V4-2*01,TCRβ V4-3*01, or TCRβ V4-1*01 M TCRβ V3 TCRβ V3-1*01 N TCRβ V2 TCRβV2*01 O TCRβ V15 TCRβ V15*01 P TCRβ V30 TCRβ V30*01, or TCRβ V30*02 QTCRβ V19 TCRβ V19*01, or TCRβ V19*02 R TCRβ V27 TCRβ V27*01. S TCRβ V28TCRβ V28*01. T TCRβ V24 TCRβ V24-1*01 U TCRβ V20 TCRβ V20-1*01, or TCRβV20-1*02 V TCRβ V25 TCRβ V25-1*01 W TCRβ V29 TCRβ V29-1*01

TABLE 8B Additional TCRβV subfamilies Subfamily TCRβ V1 TCRβ V17 TCRβV21 TCRβ V23 TCRβ V26

Exemplary amino acid sequences for TCRβV subfamily members can be foundon the ImMunoGeneTics Information System website: www.imgt.org/, or in asimilar resource.

The alignment of TCRBV amino acid sequences in Table 9 underscores thediversity of TCR sequences. In particular, the TRBV sequences fromdifferent subfamilies are considerably different from each other.

Anti-TCRβV Antibodies

Disclosed herein, is the discovery of a novel class of antibodies, i.e.anti-TCRBV antibody molecules disclosed herein, which despite having lowsequence similarity (e.g., low sequence identity among the differentantibody molecules that recognize different TCRBV subfamilies),recognize a structurally conserved region, e.g., domain, on the TCRBVprotein (e.g., as denoted by the circled area in FIG. 24A) and have asimilar function (e.g., a similar cytokine profile). Thus, theanti-TCRBV antibody molecules disclosed herein share astructure-function relationship.

Without wishing to be bound by theory, it is believed that in someembodiments, the anti-TCRβV antibody molecules disclosed herein bind toan outward facing epitope of a TCRβV protein when it is in a complexwith a TCRalpha protein, e.g., as described by the circled area in FIG.24A. In some embodiments, the anti-TCRβV antibody molecules disclosedherein recognize (e.g., bind to), a structurally conserved domain on theTCRβV protein (e.g., as denoted by the circled area in FIG. 24A).

In some embodiments, the anti-TCRβV antibody molecules disclosed hereindo not recognize, e.g., bind to, an interface of a TCRβV:TCRalphacomplex.

In some embodiments, the anti-TCRβV antibody molecules disclosed hereindo not recognize, e.g., bind to, a constant region of a TCRβV protein.An exemplary antibody that binds to a constant region of a TCRBV regionis JOVI. 1 as described in Viney et al., (Hybridoma. 1992 December;11(6):701-13).

In some embodiments, the anti-TCRβV antibody molecules disclosed hereindo not recognize, e.g., bind to, one or more (e.g., all) of acomplementarity determining region (e.g., CDR1, CDR2 and/or CDR3) of aTCRβV protein.

In some embodiments, the anti-TCRβV antibody molecules disclosed hereinbinds (e.g., specifically binds) to a TCRβV region. In some embodiments,binding of anti-TCRβV antibody molecules disclosed herein results in acytokine profile that differs from a cytokine profile of a T cellengager that binds to a receptor or molecule other than a TCRβV region(“a non-TCRBV-binding T cell engager”). In some embodiments, thenon-TCRβV-binding T cell engager comprises an antibody that binds to aCD3 molecule (e.g., CD3 epsilon (CD3e) molecule); or a TCR alpha (TCRa)molecule. In some embodiments, the non-TCRβV-binding T cell engager isan OKT3 antibody or an SP34-2 antibody.

In an aspect, the disclosure provides an anti-TCRβV antibody moleculethat binds to human TCRβV, e.g., a TCRβV gene family, e.g., one or moreof a TCRβV subfamily, e.g., as described herein, e.g., in FIG. 3, Table8A, or Table 8B. In some embodiments, the anti-TCRβV antibody moleculebinds to one or more TCRβV subfamilies chosen from: a TCRβ V6 subfamily,a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5 subfamily, a TCRβV7 subfamily, a TCRβ V11 subfamily, a TCRβ V14 subfamily, a TCRβ V16subfamily, a TCRβ V18 subfamily, a TCRβ V9 subfamily, a TCRβ V13subfamily, a TCRβ V4 subfamily, a TCRβ V3 subfamily, a TCRβ V2subfamily, a TCRβ V15 subfamily, a TCRβ V30 subfamily, a TCRβ V19subfamily, a TCRβ V27 subfamily, a TCRβ V28 subfamily, a TCRβ V24subfamily, a TCRβ V20 subfamily, TCRβ V25 subfamily, a TCRβ V29subfamily, a TCRβ V1 subfamily, a TCRβ V17 subfamily, a TCRβ V21subfamily, a TCRβ V23 subfamily, or a TCRβ V26 subfamily, or a variantthereof.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβ V6subfamily comprising: TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβV6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβV6-3*01 or TCR3 V6-1*01, or a variant thereof. In some embodiments theTCRβ V6 subfamily comprises TCRβ V6-5*01, or a variant thereof. In someembodiments, TCRβ V6 comprises TCRβ V6-4*01, or a variant thereof. Insome embodiments, TCRβ V6 comprises TCRβ V6-4*02, or a variant thereof.In some embodiments, TCRβ V6 comprises TCRβ V6-9*01, or a variantthereof. In some embodiments, TCRβ V6 comprises TCRβ V6-8*01, or avariant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-5*01, ora variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-6*02,or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβV6-6*01, or a variant thereof. In some embodiments, TCRβ V6 comprisesTCRβ V6-2*01, or a variant thereof. In some embodiments, TCRβ V6comprises TCRβ V6-3*01, or a variant thereof. In some embodiments, TCRβV6 comprises TCRβ V6-1*01, or a variant thereof.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβV10 subfamily comprising: TCRβ V10-1*01, TCRβ V10-1*02, TCRβ V10-3*01 orTCRβ V10-2*01, or a variant thereof.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβV12 subfamily comprising: TCRβ V12-4*01, TCRβ V12-3*01 or TCRβ V12-5*01,or a variant thereof.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβ V5subfamily comprising: TCRβ V5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβV5-8*01, TCRβ V5-1*01, or a variant thereof.

Exemplary anti-TCRβV antibody molecules and the corresponding TCRβVsubfamily recognized by said anti-TCRβV antibody molecules is disclosedin Table 13.

TABLE 13 Exemplary anti-TCRβV antibody molecules TRBV TRBV Reagentsmonoclonal antibodies gene name allele name Clone name and SpecificityCompany product Isotype TRBV2 TRBV2*01 IsMMU 546 (TRBV2) Serotec V BETA22 Mouse TRBV2*02 Coulter Vbeta22 IgG1 TRBV2*03 TRBV3-1 TRBV3-1*01 FIN9(TRBV3-1) Serotec Vbeta9 Mouse AMKB1-2 (TRBV3-1) Coulter Vbeta9 IgG2aTRBV3-1*02 BD Biosciences Mouse Vbeta9 IgG1 TRBV4-1 TRBV4-1*01 ZOE(TRBV4-1, TRBV4-2, Serotec V BETA 7 Mouse TRBV4-3) Coulter Vbeta7 IgG2aTRBV4-1*02 3G5 (TRBV4-1) Pierce EndogenV beta Mouse 7.1 IgG2b TRBV4-2TRBV4-2*01 ZOE (TRBV4-1, TRBV4-2, Serotec V BETA 7 Mouse TRBV4-2*02TRBV4-3) Coulter Vbeta7 IgG2a TRBV4-3 TRBV4-3*01 ZOE (TRBV4-1, TRBV4-2,Serotec V BETA 7 Mouse TRBV4-3*02 TRBV4-3) Coulter Vbeta7 IgG2aTRBV4-3*03 TRBV4-3*04 ZIZOU4 (TRBV4-3) Coulter Vbeta7.2 Mouse IgG2aTRBV5-1 TRBV5-1*01 IMMU157 (TRBV5-1) Serotec Vbeta5.1 Mouse CoulterVbeta5.1 IgG2a TRBV5-1*02 LC4 (TRBV5-1) Pierce Endogen V beta Mouse 5(c)IgG1 BD Biosciences Vbeta5(c) TRBV5-4 TRBV5-4*01 TRBV5-4*02 TRBV5-4*03TRBV5-4*04 TRBV5-5 TRBV5-5*01 3D11 (TRBV5-5) Serotec VBETA5.3 Mouse 1C1(TRBV5-5, TRBV5-6) Coulter Vbeta5.3 IgG1 TRBV5-5*02 W112 (TRBV5-5)Pierce Endogen V beta Mouse MH3-2 (TRBV5-5, 5(a) IgG1 TRBV5-6) BDBiosciences 4H11 (TM27) as disclosed in Vbeta5(a) TRBV5-5*03 U.S. Pat.No. 5,861,155 Pierce Endogen V beta Mouse 5(b) IgG1 Serotec V beta5.2/5.3 BD Biosciences Vbeta5(b) BD Biosciences Mouse Vbeta5 IgG2aTRBV5-6 TRBV5-6*01 36213 (TRBV5-6) Serotec Vbeta5.2 Mouse 1C1 (TRBV5-5,TRBV5-6) BD Biosciences IgG1 MH3-2 (TRBV5-5, TRBV5-6) Vbeta5(a) Mouse BDBiosciences IgG1 Vbeta5 Mouse IgG2a TRBV5-8 TRBV5-8*01 TRBV5-8*02TRBV6-1 TRBV6-1*01 BAM13 (TRBV6-1, Pierce Endogen V beta Mouse TRBV6-5)13 IgG1 BD Biosciences Vbeta13.1, 13.3 TRBV6-2 TRBV6-2*01 H132 CoulterVbeta13.2 Mouse IgG1 TRBV6-3 TRBV6-3*01 TRBV6-4 TRBV6-4*01 TRBV6-4*02TRBV6-5 TRBV6-5*01 IMMU 222 (TRBV6-5, Serotec V BETA 13.1 Mouse TRBV6-6and TRBV6-9) Coulter Vbeta13.1 IgG2b BAM13 (TRBV6-1, Pierce Endogen Vbeta Mouse TRBV6-5) 13 IgG1 BD Biosciences Vbeta13.1, 13.3 TRBV6-6TRBV6-6*01 JU-74 (TRBV6-6) Serotec Vbeta13.6 Mouse TRBV6-6*02 JU74.3(TRBV6-6) Coulter Vbeta13.6 IgG1 TRBV6-6*03 IMMU 222 (TRBV6-5, Serotec VBETA 13.1 Mouse TRBV6-6*04 TRBV6-6 and TRBV6-9) Coulter Vbeta13.1 IgG2bTRBV6-6*05 TRBV6-8 TRBV6-8*01 TRBV6-9 TRBV6-9*01 IMMU 222 (TRBV6-5,Serotec V BETA 13.1 Mouse TRBV6-6 and TRBV6-9) Coulter Vbeta13.1 IgG2bTRBV7-2 TRBV7-2*01 OT145 (TRBV7-2) Pierce Endogen V beta 6.7 MouseTRBV7-2*02 BD Biosciences IgG1 TRBV7-2*03 Vbeta6.7 TRBV7-2*04 TRBV7-3TRBV7-3*01 TRBV7-3*04 TRBV7-3*05 TRBV7-4 TRBV7-4*01 TRBV7-6 TRBV7-6*01TRBV7-6*02 TRBV7-7 TRBV7-7*01 TRBV7-7*02 TRBV7-8 TRBV7-8*01 TRBV7-8*02TRBV7-8*03 TRBV7-9 TRBV7-9*01 TRBV7-9*02 TRBV7-9*03 TRVB7-9*04TRBV7-9*05 TRBV7-9*06 TRBV7-9*07 TRBV9 TRBV9*01 BL37.2 (TRBV9) SerotecVbeta1 Rat IgG1 TRBV9*02 Coulter Vbeta1 TRBV9*03 TRBV10-1 TRBV10-1*01S511 (TRBV10-1, Pierce Endogen V beta 12 Mouse TRBV10-1*02 TRBV10-2,TRBV10-3) BD Biosciences IgG2b TRBV10-2 TRBV10-2*01 Vbeta12 TRBV10-2*02TRBV10-3 TRBV10-3*01 VER2.32.1 (TRBV10-3) Serotec Vbeta12 MouseTRBV10-3*02 S511 (TRBV10-1, Coulter Vbeta12 IgG2a TRBV10-3*03 TRBV10-2,TRBV10-3) Pierce Endogen V beta 12 Mouse TRBV10-3*04 BD BiosciencesIgG2b Vbeta12 TRBV11-1 TRBV11-1*01 TRBV11-2 TRBV11-2*01 IG125 (TRBV11-2)Serotec Vbeta21.3 Mouse TRBV11-2*02 Coulter Vbeta21.3 IgG2a TRBV11-2*03TRBV11-3 TRBV11-3*01 TRBV11-3*02 TRBV11-3*03 TRBV11-3*04 TRBV12-3TRBV12-3*01 56C5 (TRBV12-3, Serotec Vbeta8.1/8.2 Mouse TRBV12-4) CoulterVbeta8 IgG2a TRBV12-4 TRBV12-4*01 56C5.2 (TRBV12-3, Pierce Endogen Vbeta Mouse TRBV12-4) 8(a) IgG2b 16G8 (TRBV12-3, BD Biosciences TRBV12-4)Vbeta8 TRBV12-4*02 MX-6 (TRBV12-3, Pierce Endogen V beta Mouse TRBV12-4)8(b) IgG2a JR2 (TRBV12-3, TRBV12- BD Biosciences Mouse 4, TRBV12-5)Vbeta8 IgG2b TRBV12-5 TRBV12-5*01 JR2 (TRBV12-3, TRBV12- BD BiosciencesMouse 4, TRBV12-5) Vbeta8 IgG2b TRBV13 TRBV13*01 AF-23 (TRBV13) SerotecVbeta23 Mouse TRBV13*02 AF23 (TRBV13) Coulter Vbeta23 IgG1 AHUT7(Vbeta23) BD Biosciences Vbeta23 TRBV14 TRBV14*01 TAMAYA1.2 (TRBV14)Serotec Vbeta16 Mouse TRBV14*02 Coulter Vbeta16 IgG1 TRBV15 TRBV15*01TRBV15*02 TRBV15*03 TRBV16 TRBV16*01 TRBV16*03 TRBV18 TRBV18*01 BA62(TRBV18) Serotec V BETA 18 Mouse BA62.6 (TRBV18) Coulter Vbeta18 IgG1TRBV19 TRBV19*01 C1 (TRBV19) Pierce Endogen V beta Mouse 17 IgG1 BDBiosciences Vbeta17 TRBV19*02 E17.5F3 (TRBV19) Serotec Vbeta17 MouseTRBV19*03 E17.5F3.15.13 (TRBV19) Coulter Vbeta17 IgG1 TRBV20-1TRBV20-1*01 MPB2D5 (TRBV20-1) Serotec VBETA2 Mouse TRBV20-1*02 CoulterVbeta2 IgG1 TRBV20-1*03 TRBV20-1*04 TRBV20-1*05 TRBV20-1*06 TRBV20-1*07TRBV24-1 TRBV24-1*01 TRBV25-1 TRBV25-1*01 C21 (TRBV25-1) Serotec V BETA11 Mouse Coulter Vbeta11 IgG2a TRBV27 TRBV27*01 CAS1.1.3 (TRBV27)Serotec Vbeta14 Mouse Coulter Vbeta14 IgG1 TRBV28 TRBV28*01 CH92(TRBV28) Serotec Vbeta3 Mouse 8F10 (TRBV28) Coulter Vbeta3 IgM JOVI-3(TRBV28) Pierce Endogen V beta Mouse 3.1 IgG1 BD Biosciences MouseVbeta3 IgG2a TRBV29-1 TRBV29-1*01 WJF24 Coulter Vbeta4 Rat IgMTRBV29-1*02 TRBV29-1*03 TRBV30 TRBV30*01 ELL1.4 (TRBV30) Serotec Vbeta20Mouse TRBV30*02 Coulter Vbeta20 IgG1 TRBV30*04 TRBV30*05

In some embodiments, the anti-TCRβV antibody molecule does not bind toTCRβ V12, or binds to TCRβ V12 with an affinity and/or bindingspecificity that is less than (e.g., less than about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/orbinding specificity of the 16G8 murine antibody or a humanized versionthereof as described in U.S. Pat. No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to TCRβ V12with an affinity and/or binding specificity that is greater than (e.g.,greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about2-, 5-, or 10-fold) the affinity and/or binding specificity of the 16G8murine antibody or a humanized version thereof as described in U.S. Pat.No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβVregion other than TCRβ V12 (e.g., TCRβV region as described herein,e.g., TCRβ V6 subfamily (e.g., TCRβ V6-5*01) with an affinity and/orbinding specificity that is greater than (e.g., greater than about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) theaffinity and/or binding specificity of the 16G8 murine antibody or ahumanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule does not bind toTCRβ V5-5*01 or TCRβ V5-1*01, or binds to TCRβ V5-5*01 or TCRβ V5-1*01with an affinity and/or binding specificity that is less than (e.g.,less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-,5-, or 10-fold) the affinity and/or binding specificity of the TM23murine antibody or a humanized version thereof as described in U.S. Pat.No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to TCRβV5-5*01 or TCRβ V5-1*01 with an affinity and/or binding specificity thatis greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/or bindingspecificity of the TM23 murine antibody or a humanized version thereofas described in U.S. Pat. No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβVregion other than TCRβ V5-5*01 or TCRβ V5-1*01 (e.g., TCRβV region asdescribed herein, e.g., TCRβ V6 subfamily (e.g., TCRβ V6-5*01) with anaffinity and/or binding specificity that is greater than (e.g., greaterthan about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-,or 10-fold) the affinity and/or binding specificity of the TM23 murineantibody or a humanized version thereof as described in U.S. Pat. No.5,861,155.

Anti-TCRβ V6 Antibodies

Accordingly, in one aspect, the disclosure provides an anti-TCRβVantibody molecule that binds to human TCRβ V6, e.g., a TCRβ V6 subfamilycomprising: TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβV6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβV6-1*01. In some embodiments the TCRβ V6 subfamily comprises TCRβV6-5*01 or a variant thereof. In some embodiments, TCRβ V6 comprisesTCRβ V6-4*01, or a variant thereof. In some embodiments, TCRβ V6comprises TCRβ V6-4*02, or a variant thereof. In some embodiments, TCRβV6 comprises TCRβ V6-9*01, or a variant thereof. In some embodiments,TCRβ V6 comprises TCRβ V6-8*01, or a variant thereof. In someembodiments, TCRβ V6 comprises TCRβ V6-5*01, or a variant thereof. Insome embodiments, TCRβ V6 comprises TCRβ V6-6*02, or a variant thereof.In some embodiments, TCRβ V6 comprises TCRβ V6-6*01, or a variantthereof. In some embodiments, TCRβ V6 comprises TCRβ V6-2*01, or avariant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-3*01, ora variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-1*01,or a variant thereof.

In some embodiments, TCRβ V6-5*01 is encoded by the nucleic acidsequence of SEQ ID NO: 43, or a sequence having 85%, 90%, 95%, 99% ormore identity thereof.

SEQ ID NO: 43 ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACT C

In some embodiments, TCRβ V6-5*01 comprises the amino acid sequence ofSEQ ID NO: 44, or an amino acid sequence having 85%, 90%, 95%, 99% ormore identity thereof.

SEQ ID NO: 44 MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLL SAAPSQTSVYFCASSY

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is a non-murine antibodymolecule, e.g., a human or humanized antibody molecule. In someembodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g.,anti-TCRβ V6-5*01) antibody molecule is a human antibody molecule. Insome embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6(e.g., anti-TCRβ V6-5*01) antibody molecule is a humanized antibodymolecule.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is isolated orrecombinant.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least oneantigen-binding region, e.g., a variable region or an antigen-bindingfragment thereof, from an antibody described herein, e.g., an antibodychosen from A-H.1 or A-H.2, or an antibody described in Table 1, orencoded by a nucleotide sequence in Table 1, or a sequence substantiallyidentical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% orhigher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one,two, three or four variable regions from an antibody described herein,e.g., an antibody chosen from A-H.1 or A-H.2, or an antibody describedin Table 1, or encoded by a nucleotide sequence in Table 1, or asequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least oneor two heavy chain variable regions from an antibody described herein,e.g., an antibody chosen from A-H.1 or A-H.2, or an antibody moleculedescribed in Table 1, or encoded by a nucleotide sequence in Table 1, ora sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In some embodiments, the anti-TCRβV antibody molecule comprises a heavychain variable region (VH) having a consensus sequence of SEQ ID NO: 231or 3290.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least oneor two light chain variable regions from an antibody described herein,e.g., an antibody chosen from A-H.1 or A-H.2, or an antibody describedin Table 1, or encoded by a nucleotide sequence in Table 1, or asequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In some embodiments, the anti-TCRβV antibody molecule comprises a lightchain variable region (VL) having a consensus sequence of SEQ ID NO: 230or 3289.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chainconstant region for an IgG4, e.g., a human IgG4. In still anotherembodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g.,anti-TCRβ V6-5*01) antibody molecule includes a heavy chain constantregion for an IgG1, e.g., a human IgG1. In one embodiment, the heavychain constant region comprises an amino sequence set forth in Table 3,or a sequence substantially identical (e.g., at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or higher identical) thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes a kappa lightchain constant region, e.g., a human kappa light chain constant region.In one embodiment, the light chain constant region comprises an aminosequence set forth in Table 3, or a sequence substantially identical(e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higheridentical) thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one,two, or three complementarity determining regions (CDRs) from a heavychain variable region (VH) of an antibody described herein, e.g., anantibody chosen from A-H.1 or A-H.2, or an antibody described in Table1, or encoded by a nucleotide sequence in Table 1, or a sequencesubstantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one,two, or three CDRs (or collectively all of the CDRs) from a heavy chainvariable region comprising an amino acid sequence shown in Table 1, orencoded by a nucleotide sequence shown in Table 1. In one embodiment,one or more of the CDRs (or collectively all of the CDRs) have one, two,three, four, five, six or more changes, e.g., amino acid substitutionsor deletions, relative to the amino acid sequence shown in Table 1, orencoded by a nucleotide sequence shown in Table 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one,two, or three complementarity determining regions (CDRs) from a lightchain variable region of an antibody described herein, e.g., an antibodychosen from A-H.1 or A-H.2, or an antibody described in Table 1, orencoded by a nucleotide sequence in Table 1, or a sequence substantiallyidentical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% orhigher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one,two, or three CDRs (or collectively all of the CDRs) from a light chainvariable region comprising an amino acid sequence shown in Table 1, orencoded by a nucleotide sequence shown in Table 1. In one embodiment,one or more of the CDRs (or collectively all of the CDRs) have one, two,three, four, five, six or more changes, e.g., amino acid substitutionsor deletions, relative to the amino acid sequence shown in Table 1, orencoded by a nucleotide sequence shown in Table 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one,two, three, four, five or six CDRs (or collectively all of the CDRs)from a heavy and light chain variable region comprising an amino acidsequence shown in Table 1, or encoded by a nucleotide sequence shown inTable 1. In one embodiment, one or more of the CDRs (or collectively allof the CDRs) have one, two, three, four, five, six or more changes,e.g., amino acid substitutions or deletions, relative to the amino acidsequence shown in Table 1, or encoded by a nucleotide sequence shown inTable 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, molecule includes allsix CDRs from an antibody described herein, e.g., an antibody chosenfrom A-H.1 or A-H.2, or an antibody described in Table 1, or encoded bya nucleotide sequence in Table 1, or closely related CDRs, e.g., CDRswhich are identical or which have at least one amino acid alteration,but not more than two, three or four alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions). In someembodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g.,anti-TCRβ V6-5*01) antibody molecule, may include any CDR describedherein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule includes at least one,two, or three CDRs according to Kabat et al. (e.g., at least one, two,or three CDRs according to the Kabat definition as set out in Table 1)from a heavy chain variable region of an antibody described herein,e.g., an antibody chosen from A-H.1 or A-H.2, or an antibody describedin Table 1, or a sequence substantially identical (e.g., at least 80%,85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of theaforesaid sequences; or which have at least one amino acid alteration,but not more than two, three or four alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) relative toone, two, or three CDRs according to Kabat et al. shown in Table 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule includes at least one,two, or three CDRs according to Kabat et al. (e.g., at least one, two,or three CDRs according to the Kabat definition as set out in Table 1)from a light chain variable region of an antibody described herein,e.g., an antibody chosen from A-H.1 or A-H.2, or an antibody describedin Table 1, or a sequence substantially identical (e.g., at least 80%,85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of theaforesaid sequences; or which have at least one amino acid alteration,but not more than two, three or four alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) relative toone, two, or three CDRs according to Kabat et al. shown in Table 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one,two, three, four, five, or six CDRs according to Kabat et al. (e.g., atleast one, two, three, four, five, or six CDRs according to the Kabatdefinition as set out in Table 1) from the heavy and light chainvariable regions of an antibody described herein, e.g., an antibodychosen from A-H.1 or A-H.2, or an antibody described in Table 1, orencoded by a nucleotide sequence in Table 1; or a sequence substantiallyidentical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% orhigher identical) to any of the aforesaid sequences; or which have atleast one amino acid alteration, but not more than two, three or fouralterations (e.g., substitutions, deletions, or insertions, e.g.,conservative substitutions) relative to one, two, three, four, five, orsix CDRs according to Kabat et al. shown in Table 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes all six CDRsaccording to Kabat et al. (e.g., all six CDRs according to the Kabatdefinition as set out in Table 1) from the heavy and light chainvariable regions of an antibody described herein, e.g., an antibodychosen from A-H.1 or A-H.2, or an antibody described in Table 1, orencoded by a nucleotide sequence in Table 1; or a sequence substantiallyidentical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% orhigher identical) to any of the aforesaid sequences; or which have atleast one amino acid alteration, but not more than two, three or fouralterations (e.g., substitutions, deletions, or insertions, e.g.,conservative substitutions) relative to all six CDRs according to Kabatet al. shown in Table 1. In one embodiment, the anti-TCRβV antibodymolecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibodymolecule, may include any CDR described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one,two, or three hypervariable loops that have the same canonicalstructures as the corresponding hypervariable loop of an antibodydescribed herein, e.g., an antibody chosen from chosen from A-H.1 orA-H.2 e.g., the same canonical structures as at least loop 1 and/or loop2 of the heavy and/or light chain variable domains of an antibodydescribed herein. See, e.g., Chothia et al., (1992) J. Mol. Biol.227:799-817; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798 fordescriptions of hypervariable loop canonical structures. Thesestructures can be determined by inspection of the tables described inthese references.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule includes at least one,two, or three CDRs according to Chothia et al. (e.g., at least one, two,or three CDRs according to the Chothia definition as set out in Table 1)from a heavy chain variable region of an antibody described herein,e.g., an antibody chosen from A-H.1 or A-H.2, or as described in Table1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences; or which have at least one amino acid alteration, but notmore than two, three or four alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) relative toone, two, or three CDRs according to Chothia et al. shown in Table 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule includes at least one,two, or three CDRs according to Chothia et al. (e.g., at least one, two,or three CDRs according to the Chothia definition as set out in Table 1)from a light chain variable region of an antibody described herein,e.g., an antibody chosen from A-H.1 or A-H.2, or an antibody describedin Table 1, or a sequence substantially identical (e.g., at least 80%,85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of theaforesaid sequences; or which have at least one amino acid alteration,but not more than two, three or four alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) relative toone, two, or three CDRs according to Chothia et al. shown in Table 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one,two, three, four, five, or six CDRs according to Chothia et al. (e.g.,at least one, two, three, four, five, or six CDRs according to theChothia definition as set out in Table 1) from the heavy and light chainvariable regions of an antibody described herein, e.g., an antibodychosen from A-H.1 or A-H.2, or an antibody described in Table 1, orencoded by the nucleotide sequence in Table 1; or a sequencesubstantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or higher identical) to any of the aforesaid sequences; orwhich have at least one amino acid alteration, but not more than two,three or four alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions) relative to one, two,three, four, five, or six CDRs according to Chothia et al. shown inTable 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes all six CDRsaccording to Chothia et al. (e.g., all six CDRs according to the Chothiadefinition as set out in Table 1) from the heavy and light chainvariable regions of an antibody described herein, e.g., an antibodychosen from A-H.1 or A-H.2, or an antibody described in Table 1, orencoded by a nucleotide sequence in Table 1; or a sequence substantiallyidentical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% orhigher identical) to any of the aforesaid sequences; or which have atleast one amino acid alteration, but not more than two, three or fouralterations (e.g., substitutions, deletions, or insertions, e.g.,conservative substitutions) relative to all six CDRs according toChothia et al. shown in Table 1. In one embodiment, the anti-TCRβVantibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibodymolecule, may include any CDR described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, molecule includes acombination of CDRs or hypervariable loops defined according to Kabat etal., Chothia et al., or as described in Table 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, can contain anycombination of CDRs or hypervariable loops according to the Kabat andChothia definitions.

In some embodiments, a combined CDR as set out in Table 1 is a CDR thatcomprises a Kabat CDR and a Chothia CDR.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, molecule includes acombination of CDRs or hypervariable loops identified as combined CDRsin Table 1. In some embodiments, the anti-TCRβV antibody molecule, e.g.,anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, can containany combination of CDRs or hypervariable loops according the “combined”CDRs are described in Table 1.

In an embodiment, e.g., an embodiment comprising a variable region, aCDR (e.g., a combined CDR, Chothia CDR or Kabat CDR), or other sequencereferred to herein, e.g., in Table 1, the antibody molecule is amonospecific antibody molecule, a bispecific antibody molecule, abivalent antibody molecule, a biparatopic antibody molecule, or anantibody molecule that comprises an antigen binding fragment of anantibody, e.g., a half antibody or antigen binding fragment of a halfantibody. In certain embodiments the antibody molecule comprises amultispecific molecule, e.g., a bispecific molecule, e.g., as describedherein.

In an embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6(e.g., anti-TCRβ V6-5*01) antibody molecule includes:

(i) one, two or all of a light chain complementarity determining region1 (LC CDR1), a light chain complementarity determining region 2 (LCCDR2), and a light chain complementarity determining region 3 (LC CDR3)of SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 11, and/or(ii) one, two or all of a heavy chain complementarity determining region1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2),and a heavy chain complementarity determining region 3 (HC CDR3) of SEQID NO: 1 or SEQ ID NO: 9.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a LC CDR1, LCCDR2, and LC CDR3 of SEQ ID NO: 2, and a HC CDR1, HC CDR2, and HC CDR3of SEQ ID NO: 1.

In some embodiments the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6(e.g., anti-TCRβ V6-5*01) antibody molecule comprises a LC CDR1, LCCDR2, and LC CDR3 of SEQ ID NO: 10, and a HC CDR1, HC CDR2, and HC CDR3of SEQ ID NO: 9.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a LC CDR1, LCCDR2, and LC CDR3 of SEQ ID NO: 11, and a HC CDR1, HC CDR2, and HC CDR3of SEQ ID NO: 9.

In an embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6(e.g., anti-TCRβ V6-5*01) antibody molecule comprises:

(i) a LC CDR1 amino acid sequence of SEQ ID NO: 6, a LC CDR2 amino acidsequence of SEQ ID NO: 7, or a LC CDR3 amino acid sequence of SEQ ID NO:8; and/or(ii) a HC CDR1 amino acid sequence of SEQ ID NO: 3, a HC CDR2 amino acidsequence of SEQ ID NO: 4, or a HC CDR3 amino acid sequence of SEQ ID NO:5.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises:

(i) a light chain variable region (VL) comprising a LC CDR1 amino acidsequence of SEQ ID NO: 6, a LC CDR2 amino acid sequence of SEQ ID NO: 7,or a LC CDR3 amino acid sequence of SEQ ID NO: 8; and/or(ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acidsequence of SEQ ID NO: 3, a HC CDR2 amino acid sequence of SEQ ID NO: 4,or a HC CDR3 amino acid sequence of SEQ ID NO: 5.

In an embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6(e.g., anti-TCRβ V6-5*01) antibody molecule comprises:

(i) a LC CDR1 amino acid sequence of SEQ ID NO: 51, a LC CDR2 amino acidsequence of SEQ ID NO: 52, or a LC CDR3 amino acid sequence of SEQ IDNO: 53; and/or(ii) a HC CDR1 amino acid sequence of SEQ ID NO: 45, a HC CDR2 aminoacid sequence of SEQ ID NO: 46, or a HC CDR3 amino acid sequence of SEQID NO: 47.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises:

(i) a light chain variable region (VL) comprising a LC CDR1 amino acidsequence of SEQ ID NO: 51, a LC CDR2 amino acid sequence of SEQ ID NO:52, or a LC CDR3 amino acid sequence of SEQ ID NO: 53; and/or(ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acidsequence of SEQ ID NO: 45, a HC CDR2 amino acid sequence of SEQ ID NO:46, or a HC CDR3 amino acid sequence of SEQ ID NO: 47.

In an embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6(e.g., anti-TCRβ V6-5*01) antibody molecule comprises:

(i) a LC CDR1 amino acid sequence of SEQ ID NO: 54, a LC CDR2 amino acidsequence of SEQ ID NO: 55, or a LC CDR3 amino acid sequence of SEQ IDNO: 56; and/or(ii) a HC CDR1 amino acid sequence of SEQ ID NO: 48, a HC CDR2 aminoacid sequence of SEQ ID NO: 49, or a HC CDR3 amino acid sequence of SEQID NO: 50.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises:

(i) a light chain variable region (VL) comprising a LC CDR1 amino acidsequence of SEQ ID NO: 54, a LC CDR2 amino acid sequence of SEQ ID NO:55, or a LC CDR3 amino acid sequence of SEQ ID NO: 56; and/or(ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acidsequence of SEQ ID NO: 48, a HC CDR2 amino acid sequence of SEQ ID NO:49, or a HC CDR3 amino acid sequence of SEQ ID NO: 50.

In one embodiment, the light or the heavy chain variable framework(e.g., the region encompassing at least FR1, FR2, FR3, and optionallyFR4) of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g.,anti-TCRβ V6-5*01) antibody molecule can be chosen from: (a) a light orheavy chain variable framework including at least 80%, 85%, 87% 90%,92%, 93%, 95%, 97%, 98%, or 100% of the amino acid residues from a humanlight or heavy chain variable framework, e.g., a light or heavy chainvariable framework residue from a human mature antibody, a humangermline sequence, or a human consensus sequence; (b) a light or heavychain variable framework including from 20% to 80%, 40% to 60%, 60% to90%, or 70% to 95% of the amino acid residues from a human light orheavy chain variable framework, e.g., a light or heavy chain variableframework residue from a human mature antibody, a human germlinesequence, or a human consensus sequence; (c) a non-human framework(e.g., a rodent framework); or (d) a non-human framework that has beenmodified, e.g., to remove antigenic or cytotoxic determinants, e.g.,deimmunized, or partially humanized. In one embodiment, the light orheavy chain variable framework region (particularly FR1, FR2 and/or FR3)includes a light or heavy chain variable framework sequence at least 70,75, 80, 85, 87, 88, 90, 92, 94, 95, 96, 97, 98, 99% identical oridentical to the frameworks of a VL or VH segment of a human germlinegene.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chainvariable domain having at least one, two, three, four, five, six, seven,ten, fifteen, twenty or more changes, e.g., amino acid substitutions ordeletions, from an amino acid sequence of A-H.1 or A-H.2, e.g., theamino acid sequence of the FR region in the entire variable region,e.g., shown in FIG. 1A, or in SEQ ID NO: 9.

Alternatively, or in combination with the heavy chain substitutionsdescribed herein, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6(e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chainvariable domain having at least one, two, three, four, five, six, seven,ten, fifteen, twenty or more amino acid changes, e.g., amino acidsubstitutions or deletions, from an amino acid sequence of A-H.1 orA-H.2. e.g., the amino acid sequence of the FR region in the entirevariable region, e.g., shown in FIG. 1B, or in SEQ ID NO: 10 or SEQ IDNO: 11.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes one, two,three, or four heavy chain framework regions shown in FIG. 1A, or asequence substantially identical thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes one, two,three, or four light chain framework regions shown in FIG. 1B, or asequence substantially identical thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the lightchain framework region 1 of A-H.1 or A-H.2, e.g., as shown in FIG. 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the lightchain framework region 2 of A-H.1 or A-H.2, e.g., as shown in FIG. 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the lightchain framework region 3 of A-H.1 or A-H.2, e.g., as shown in FIG. 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the lightchain framework region 4 of A-H.1 or A-H.2, e.g., as shown in FIG. 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chainvariable domain comprising a framework region, e.g., framework region 1(FR1), comprising a change, e.g., a substitution (e.g., a conservativesubstitution) at position 10 according to Kabat numbering. In someembodiments, the FR1 comprises a Phenylalanine at position 10, e.g., aSerine to Phenyalanine substitution. In some embodiments, thesubstitution is relative to a human germline light chain frameworkregion sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chainvariable domain comprising a framework region, e.g., framework region 2(FR2), comprising a change, e.g., a substitution (e.g., a conservativesubstitution) at a position disclosed herein according to Kabatnumbering. In some embodiments, FR2 comprises a Histidine at position36, e.g., a substitution at position 36 according to Kabat numbering,e.g., a Tyrosine to Histidine substitution. In some embodiments, FR2comprises an Alanine at position 46, e.g., a substitution at position 46according to Kabat numbering, e.g., an Arginine to Alanine substitution.In some embodiments, the substitution is relative to a human germlinelight chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chainvariable domain comprising a framework region, e.g., framework region 3(FR3), comprising a change, e.g., a substitution (e.g., a conservativesubstitution) at a position disclosed herein according to Kabatnumbering. In some embodiments, FR3 comprises a Phenyalanine at position87, e.g., a substitution at position 87 according to Kabat numbering,e.g., a Tyrosine to Phenyalanine substitution. In some embodiments, thesubstitution is relative to a human germline light chain frameworkregion sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chainvariable domain comprising: (a) a framework region 1 (FR1) comprising aPhenylalanine at position 10, e.g., a substitution at position 10according to Kabat numbering, e.g., a Serine to Phenyalaninesubstitution; (b) a framework region 2 (FR2) comprising a Histidine atposition 36, e.g., a substitution at position 36 according to Kabatnumbering, e.g., a Tyrosine to Histidine substitution, and a Alanine atposition 46, e.g., a substitution at position 46 according to Kabatnumbering, e.g., a Arginine to Alanine substitution; and (c) a frameworkregion 3 (FR3) comprising a Phenylalanine at position 87, e.g., asubstitution at position 87 according to Kabat numbering, e.g., aTyrosine to Phenyalanine substitution, e.g., as shown in the amino acidsequence of SEQ ID NO: 10. In some embodiments, the substitution isrelative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chainvariable domain comprising: (a) a framework region 2 (FR2) comprising aHistidine at position 36, e.g., a substitution at position 36 accordingto Kabat numbering, e.g., a Tyrosine to Histidine substitution, and aAlanine at position 46, e.g., a substitution at position 46 according toKabat numbering, e.g., a Arginine to Alanine substitution; and (b) aframework region 3 (FR3) comprising a Phenylalanine at position 87,e.g., a substitution at position 87 according to Kabat numbering, e.g.,a Tyrosine to Phenyalanine substitution, e.g., as shown in the aminoacid sequence of SEQ ID NO: 11. In some embodiments, the substitution isrelative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chainvariable domain comprising: (a) a framework region 1 (FR1) comprising achange, e.g., a substitution (e.g., a conservative substitution) at oneor more (e.g., all) positions disclosed herein according to Kabatnumbering, (b) a framework region 2 (FR2) comprising a change, e.g., asubstitution (e.g., a conservative substitution) at one or more (e.g.,all) position disclosed herein according to Kabat numbering and (c) aframework region 3 (FR3) comprising a change, e.g., a substitution(e.g., a conservative substitution) at one or more (e.g., all) positiondisclosed herein according to Kabat numbering. In some embodiments, thesubstitution is relative to a human germline light chain frameworkregion sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavychain framework region 1 of A-H.1 or A-H.2, e.g., as shown in FIG. 1A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavychain framework region 2 of A-H.1 or A-H.2, e.g., as shown in FIG. 1A

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavychain framework region 3 of A-H.1 or A-H.2, e.g., as shown in FIG. 1A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavychain framework region 4 of A-H.1 or A-H.2, e.g., as shown in FIG. 1A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chainvariable domain comprising a framework region, e.g., framework region 3(FR3), comprising a change, e.g., a substitution (e.g., a conservativesubstitution) at a position disclosed herein according to Kabatnumbering. In some embodiments, FR3 comprises a Threonine at position73, e.g., a substitution at position 73 according to Kabat numbering,e.g., a Glutamic Acid to Threonine substitution. In some embodiments,FR3 comprises a Glycine at position 94, e.g., a substitution at position94 according to Kabat numbering, e.g., an Arginine to Glycinesubstitution. In some embodiments, the substitution is relative to ahuman germline heavy chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chainvariable domain comprising a framework region 3 (FR3) comprising aThreonine at position 73, e.g., a substitution at position 73 accordingto Kabat numbering, e.g., a Glutamic Acid to Threonine substitution, anda Glycine at position 94, e.g., a substitution at position 94 accordingto Kabat numbering, e.g., a Arginine to Glycine substitution, e.g., asshown in the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavychain framework regions 1-4 of A-H.1 or A-H.2, e.g., SEQ ID NO: 9, or asshown in FIGS. 1A and 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the lightchain framework regions 1-4 of A-H.1, e.g., SEQ ID NO: 10, or as shownin FIGS. 1A and 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the lightchain framework regions 1-4 of A-H.2, e.g., SEQ ID NO: 11, or as shownin FIGS. 1A and 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavychain framework regions 1-4 of A-H.1, e.g., SEQ ID NO: 9; and the lightchain framework regions 1-4 of A-H.1, e.g., SEQ ID NO: 10, or as shownin FIGS. 1A and 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavychain framework regions 1-4 of A-H.2, e.g., SEQ ID NO: 9; and the lightchain framework regions 1-4 of A-H.2, e.g., SEQ ID NO: 11, or as shownin FIGS. 1A and 1B.

In some embodiments, the heavy or light chain variable domain, or both,of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβV6-5*01) antibody molecule, includes an amino acid sequence, which issubstantially identical to an amino acid disclosed herein, e.g., atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical to avariable region of an antibody described herein, e.g., an antibodychosen from A-H.1 or A-H.2, or as described in Table 1, or encoded bythe nucleotide sequence in Table 1; or which differs at least 1 or 5residues, but less than 40, 30, 20, or 10 residues, from a variableregion of an antibody described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one,two, three, or four antigen-binding regions, e.g., variable regions,having an amino acid sequence as set forth in Table 1, or a sequencesubstantially identical thereto (e.g., a sequence at least about 85%,90%, 95%, 99% or more identical thereto, or which differs by no morethan 1, 2, 5, 10, or 15 amino acid residues from the sequences shown inTable 1. In another embodiment, the anti-TCRβV antibody molecule, e.g.,anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule includes a VHand/or VL domain encoded by a nucleic acid having a nucleotide sequenceas set forth in Table 1, or a sequence substantially identical thereto(e.g., a sequence at least about 85%, 90%, 95%, 99% or more identicalthereto, or which differs by no more than 3, 6, 15, 30, or 45nucleotides from the sequences shown in Table 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 9, an aminoacid sequence at least about 85%, 90%, 95%, 99% or more identical to theamino acid sequence of SEQ ID NO: 9, or an amino acid sequence whichdiffers by no more than 1, 2, 5, 10, or 15 amino acid residues from theamino acid sequence of SEQ ID NO: 9; and/or

a VL domain comprising the amino acid sequence of SEQ ID NO: 10, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 10, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 9, an aminoacid sequence at least about 85%, 90%, 95%, 99% or more identical to theamino acid sequence of SEQ ID NO: 9, or an amino acid sequence whichdiffers by no more than 1, 2, 5, 10, or 15 amino acid residues from theamino acid sequence of SEQ ID NO: 9; and/or

a VL domain comprising the amino acid sequence of SEQ ID NO: 11, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 11, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule is a full antibody orfragment thereof (e.g., a Fab, F(ab′)₂, Fv, or a single chain Fvfragment (scFv)). In embodiments, the anti-TCRβV antibody molecule,e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule is amonoclonal antibody or an antibody with single specificity. In someembodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g.,anti-TCRβ V6-5*01) antibody molecule, can also be a humanized, chimeric,camelid, shark, or an in vitro-generated antibody molecule. In someembodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g.,anti-TCRβ V6-5*01) antibody molecule, is a humanized antibody molecule.The heavy and light chains of the anti-TCRβV antibody molecule, e.g.,anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, can befull-length (e.g., an antibody can include at least one, and preferablytwo, complete heavy chains, and at least one, and preferably two,complete light chains) or can include an antigen-binding fragment (e.g.,a Fab, F(ab′)2, Fv, a single chain Fv fragment, a single domainantibody, a diabody (dAb), a bivalent antibody, or bispecific antibodyor fragment thereof, a single domain variant thereof, or a camelidantibody).

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is in the form of amultispecific molecule, e.g., a bispecific molecule, e.g., as describedherein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, has a heavy chainconstant region (Fc) chosen from, e.g., the heavy chain constant regionsof IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In someembodiments, the Fc region is chosen from the heavy chain constantregions of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fcregion is chosen from the heavy chain constant region of IgG1 or IgG2(e.g., human IgG1, or IgG2). In some embodiments, the heavy chainconstant region is human IgG1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule, has a light chainconstant region chosen from, e.g., the light chain constant regions ofkappa or lambda, preferably kappa (e.g., human kappa). In oneembodiment, the constant region is altered, e.g., mutated, to modify theproperties of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6(e.g., anti-TCRβ V6-5*01) antibody molecule (e.g., to increase ordecrease one or more of: Fc receptor binding, antibody glycosylation,the number of cysteine residues, effector cell function, or complementfunction). For example, the constant region is mutated at positions 296(M to Y), 298 (S to T), 300 (T to E), 477 (H to K) and 478 (N to F) toalter Fc receptor binding (e.g., the mutated positions correspond topositions 132 (M to Y), 134 (S to T), 136 (T to E), 313 (H to K) and 314(N to F) of SEQ ID NOs: 212 or 214; or positions 135 (M to Y), 137 (S toT), 139 (T to E), 316 (H to K) and 317 (N to F) of SEQ ID NOs: 215, 216,217 or 218), e.g., relative to human IgG1.

Antibody A-H.1 comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 3278 and a light chain comprising the amino acidsequence of SEQ ID NO: 72. Antibody A-H.2 comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 3278 and a light chaincomprising the amino acid sequence of SEQ ID NO: 3279.

TABLE 1Amino acid and nucleotide sequences for murine, chimeric and humanized antibodymolecules. The antibody molecules include murine mAb Antibody A, and humanized mAbAntibody A-H Clones A-H.1 and A-H.2. The amino acid the heavy and light chain CDRs, andthe amino acid and nucleotide sequences of the heavy and light chain variable regions, and theheavy and light chains are shown. Antibody A (murine) SEQ ID NO: 3HC CDR1 (Combined) GYSFTTYYIH SEQ ID NO: 4 HC CDR2 (Combined)WFFPGSGNIKYNEKFKG SEQ ID NO: 5 HC CDR3 (Combined) SYYSYDVLDYSEQ ID NO: 45 HC CDR1 (Kabat) TYYIH SEQ ID NO: 46 HC CDR2 (Kabat)WFFPGSGNIKYNEKFKG SEQ ID NO: 47 HC CDR3 (Kabat) SYYSYDVLDY SEQ ID NO: 48HC CDR1 (Chothia) GYSFTTY SEQ ID NO: 49 HC CDR2 (Chothia) FPGSGNSEQ ID NO: 50 HC CDR3 (Chothia) SYYSYDVLDY SEQ ID NO: 1 VHQVQLQQSGPELVKPGTSVKISCKASGYSFTTYYIH WVKQRPGQGLEWIGWFFPGSGNIKYNEKFKGKATLTADTSSSTAYMQLSSLTSEESAVYFCAGSYYSY DVLDYWGHGTTLTVSS SEQ ID NO: 6LC CDR1 (Combined) KASQNVGINVV SEQ ID NO: 7 LC CDR2 (Combined) SSSHRYSSEQ ID NO: 8 LC CDR3 (Combined) QQFKSYPLT SEQ ID NO: 51 LC CDR1 (Kabat)KASQNVGINVV SEQ ID NO: 52 LC CDR2 (Kabat) SSSHRYS SEQ ID NO: 53LC CDR3 (Kabat) QQFKSYPLT SEQ ID NO: 54 LC CDR1 (Chothia) KASQNVGINVVSEQ ID NO: 55 LC CDR2 (chothia) SSSHRYS SEQ ID NO: 56 LC CDR3 (chothia)QQFKSYPLT SEQ ID NO: 2 VL DILMTQSQKFMSTSLGDRVSVSCKASQNVGINVVWHQQKPGQSPKALIYSSSHRYSGVPDRFTGSGSG TDFTLTINNVQSEDLAEYFCQQFKSYPLTFGAGTKLELK Antibody A humanized (A-H antibody) A-H.1 antibody SEQ ID NO: 3HC CDR1 (Combined)  GYSFTTYYIH SEQ ID NO: 4 HC CDR2 (Combined) WFFPGSGNIKYNEKFKG SEQ ID NO: 5 HC CDR3 (Combined)  SYYSYDVLDYSEQ ID NO: 9 VH QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS SEQ ID NO: 12 DNA VHCAGGTGCAGCTGGTTCAGTCTGGCGCCGAAGTG AAGAAACCTGGCTCCTCCGTGAAGGTGTCCTGCAAGGCTTCCGGCTACTCCTTCACCACCTACTAC ATCCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAATGGATGGGCTGGTTCTTCCCCGGCTC CGGCAACATCAAGTACAACGAGAAGTTCAAGGGCCGCGTGACCATCACCGCCGACACCTCTACCT CTACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCCGGCT CCTACTACTCTTACGACGTGCTGGATTACTGGGGCCAGGGCACCACAGTGACAGTGTCCTCT SEQ ID NO: 69 VH-IgM constant deltaMETDTLLLWVLLLWVPGSTGQVQLVQSGAEVKK CDC PGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAY MELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCL AQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNK EKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAE AKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLT KSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVT HTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPE KYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSL VMSDTAGTCY SEQ ID NO: 70 VH-IgGA1METDTLLLWVLLLWVPGSTGQVQLVQSGAEVKK PGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAY MELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSASPTSPKVFPLSLCSTQPDGNVVIACLVQ GFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDV TVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKS AVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEE LALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKK GDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY SEQ ID NO: 71 VH-IgGA2 METDTLLLWVLLLWVPGSTGQVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLE WMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGT TVTVSSASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDL YTTSSQLTLPATQCPDGKSVTCHVKHYTNSSQDVTVPCRVPPPPPCCHPRLSLHRPALEDLLLGSEANL TCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKT PLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASR QEPSQGTTTYAVTSILRVAAEDWKKGETFSCMVGHEALPLAFTQKTIDRMAGKPTHINVSVVMAEADG TCY SEQ ID NO: Heavy chainMETDTLLLWVLLLWVPGSTGQVQLVQSGAEVKK 3278 PGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAY MELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 6 LC CDR1 (Combined) KASQNVGINVV SEQ ID NO: 7LC CDR2 (Combined)) SSSHRYS SEQ ID NO: 8 LC CDR3 (Combined) QQFKSYPLTSEQ ID NO: 10 VL DIQMTQSPSFLSASVGDRVTITCKASQNVGINVVWHQQKPGKAPKALIYSSSHRYSGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 13 DNA VL GACATCCAGATGACCCAGTCTCCATCCTTCCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACA TGCAAGGCCTCTCAGAACGTGGGCATCAACGTCGTGTGGCACCAGCAGAAGCCTGGCAAGGCTCC TAAGGCTCTGATCTACTCCTCCAGCCACCGGTACTCTGGCGTGCCCTCTAGATTTTCCGGCTCTGGC TCTGGCACCGAGTTTACCCTGACAATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTTTTGC CAGCAGTTCAAGAGCTACCCTCTGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAG SEQ ID NO: 72 VL and kappa constantMETDTLLLWVLLLWVPGSTGDIQMTQSPSFLSAS region/light chainVGDRVTITCKASQNVGINVVWHQQKPGKAPKALI YSSSHRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGECA-H.2 antibody SEQ ID NO: 3 HC CDR1 (Combined) GYSFTTYYIH SEQ ID NO: 4HC CDR2 (Combined) WFFPGSGNIKYNEKFKG SEQ ID NO: 5 HC CDR3 (Combined)SYYSYDVLDY SEQ ID NO: 9 VH QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS SEQ ID NO: 12 DNA VHCAGGTGCAGCTGGTTCAGTCTGGCGCCGAAGTG AAGAAACCTGGCTCCTCCGTGAAGGTGTCCTGCAAGGCTTCCGGCTACTCCTTCACCACCTACTAC ATCCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAATGGATGGGCTGGTTCTTCCCCGGCTC CGGCAACATCAAGTACAACGAGAAGTTCAAGGGCCGCGTGACCATCACCGCCGACACCTCTACCT CTACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCCGTGTACTACTGCGCCGGCT CCTACTACTCTTACGACGTGCTGGATTACTGGGGCCAGGGCACCACAGTGACAGTGTCCTCT SEQ ID NO: Heavy chainMETDTLLLWVLLLWVPGSTGQVQLVQSGAEVKK 3278 PGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAY MELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 6 LC CDR1 (Combined) KASQNVGINVV SEQ ID NO: 7LC CDR2 (Combined)) SSSHRYS SEQ ID NO: 8 LC CDR3 (Combined) QQFKSYPLTSEQ ID NO: 11 VL DIQMTQSPSSLSASVGDRVTITCKASQNVGINVVWHQQKPGKVPKALIYSSSHRYSGVPSRFSGSGSG TDFTLTISSLQPEDVATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 14 DNA VL GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACA TGCAAGGCCTCTCAGAACGTGGGCATCAACGTCGTGTGGCACCAGCAGAAACCTGGCAAGGTGCC CAAGGCTCTGATCTACTCCTCCAGCCACAGATACTCCGGCGTGCCCTCTAGATTCTCCGGCTCTGG CTCTGGCACCGACTTTACCCTGACAATCTCCAGCCTGCAGCCTGAGGACGTGGCCACCTACTTTTG CCAGCAGTTCAAGAGCTACCCTCTGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAG SEQ ID NO: Light chainMETDTLLLWVLLLWVPGSTGDIQMTQSPSSLSAS 3279VGDRVTITCKASQNVGINVVWHQQKPGKVPKALI YSSSHRYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYFCQQFKSYPLTFGQGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGECA-H.3 antibody SEQ ID NO: 80 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRVSPGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVEDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRF SGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 81 VL DIQMTQSPSFLSASVGDRVTITCKASQNVEDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 82 VH QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRVSPGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.4 SEQ ID NO: 83 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VEDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTF GQGTKLEIK SEQ ID NO: 84 VLDIQMTQSPSFLSASVGDRVTITCKASQNVEDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 85 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.5SEQ ID NO: 86 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGHDFRDFYIHWVRQAPGQGLEWMGRVYPGSGSYRYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 87 VL DIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 88 VH QVQLVQSGAEVKKPGSSVKVSCKASGHDFRDFYIHWVRQAPGQGLEWMGRVYPGSGSYRYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.6 SEQ ID NO: 89 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGHDFKLTYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VDNRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 90 VLDIQMTQSPSFLSASVGDRVTITCKASQNVDNRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 91 VHQVQLVQSGAEVKKPGSSVKVSCKASGHDFKLTYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.7SEQ ID NO: 92 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVENKVAWHQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 93 VL DIQMTQSPSFLSASVGDRVTITCKASQNVENKVAWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 94 VH QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.8 SEQ ID NO: 95 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRIFAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 96 VLDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 97 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRIFAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.9SEQ ID NO: 98 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKFYIHWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVGNRVAWYQQKPGKAPKALIYSSSHRYSGVPSRF SGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 99 VL DIQMTQSPSFLSASVGDRVTITCKASQNVGNRVAWYQQKPGKAPKALIYSSSHRYSGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 100 VH QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKFYIHWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.10 SEQ ID NO: 101 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGHDFDKFYI HWVRQAPGQGLEWMGRIFAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VGDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIKs SEQ ID NO: 102 VLDIQMTQSPSFLSASVGDRVTITCKASQNVGDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 103 VHQVQLVQSGAEVKKPGSSVKVSCKASGHDFDKFYI HWVRQAPGQGLEWMGRIFAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.11SEQ ID NO: 104 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRVSPGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVGDRVAWYQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 105 VL DIQMTQSPSFLSASVGDRVTITCKASQNVGDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 106 VH QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRVSPGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.12 SEQ ID NO: 107 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRVSAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VGNRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 108 VLDIQMTQSPSFLSASVGDRVTITCKASQNVGNRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 109 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRVSAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.13SEQ ID NO: 110 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVDNRVAWYQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 111 VL DIQMTQSPSFLSASVGDRVTITCKASQNVDNRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 112 VH QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.14 SEQ ID NO: 113 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRISAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 114 VLDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 115 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRISAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.15SEQ ID NO: 116 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTYIHWVRQAPGQGLEWMGRVSPGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVDNKVAWHQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 117 VL DIQMTQSPSFLSASVGDRVTITCKASQNVDNKVAWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 118 VH QVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTYIHWVRQAPGQGLEWMGRVSPGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.16 SEQ ID NO: 119 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGGTFRLTYI HWVRQAPGQGLEWMGRVYPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 120 VLDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 121 VHQVQLVQSGAEVKKPGSSVKVSCKASGGTFRLTYI HWVRQAPGQGLEWMGRVYPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.17SEQ ID NO: 122 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTYIHWVRQAPGQGLEWMGRIFPGSGNTKYNEKFKGR VTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGS GGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFS GSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 123 VL DIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 124 VH QVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTYIHWVRQAPGQGLEWMGRIFPGSGNTKYNEKFKGR VTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.18 SEQ ID NO: 125 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYI HWVRQAPGQGLEWMGRIFPGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VEDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTF GQGTKLEIK SEQ ID NO: 126 VLDIQMTQSPSFLSASVGDRVTITCKASQNVEDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 127 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYI HWVRQAPGQGLEWMGRIFPGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.19SEQ ID NO: 128 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGGTFRLTYIHWVRQAPGQGLEWMGRISAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVGDRVAWYQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 129 VL DIQMTQSPSFLSASVGDRVTITCKASQNVGDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 130 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFRLTYIHWVRQAPGQGLEWMGRISAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.20 SEQ ID NO: 131 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGGTFDKTYI HWVRQAPGQGLEWMGRISAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 132 VLDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 133 VHQVQLVQSGAEVKKPGSSVKVSCKASGGTFDKTYI HWVRQAPGQGLEWMGRISAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.21SEQ ID NO: 134 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKFYIHWVRQAPGQGLEWMGRISAGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 135 VL DIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 136 VH QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKFYIHWVRQAPGQGLEWMGRISAGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.22 SEQ ID NO: 137 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VDNKVAWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 138 VLDIQMTQSPSFLSASVGDRVTITCKASQNVDNKVA WHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 139 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.23SEQ ID NO: 140 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTYIHWVRQAPGQGLEWMGRISAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVADRVAWYQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 141 VL DIQMTQSPSFLSASVGDRVTITCKASQNVADRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 142 VH QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTYIHWVRQAPGQGLEWMGRISAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.24 SEQ ID NO: 143 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGHDFHLWY IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQ NVDNKVAWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPL TFGQGTKLEIK SEQ ID NO: 144 VLDIQMTQSPSFLSASVGDRVTITCKASQNVDNKVA WHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 145 VHQVQLVQSGAEVKKPGSSVKVSCKASGHDFHLWY IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS YYSYDVLDYWGQGTTVTVSS A-H.25SEQ ID NO: 146 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGHDFHLWYIHWVRQAPGQGLEWMGRVFAGSGNTKYNEKFK GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVEDKVAWYQQKPGKAPKALIYSSSHRYKGVPS RFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 147 VL DIQMTQSPSFLSASVGDRVTITCKASQNVEDKVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 148 VH QVQLVQSGAEVKKPGSSVKVSCKASGHDFHLWYIHWVRQAPGQGLEWMGRVFAGSGNTKYNEKFK GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.26 SEQ ID NO: 149 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYI HWVRQAPGQGLEWMGRIFPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYS YDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNV DDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFG QGTKLEIK SEQ ID NO: 150 VLDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 151 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYI HWVRQAPGQGLEWMGRIFPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYS YDVLDYWGQGTTVTVSS A-H.27SEQ ID NO: 153 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVGNRVAWYQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 154 VL DIQMTQSPSFLSASVGDRVTITCKASQNVGNRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 155 VH QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.28 SEQ ID NO: 156 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYI HWVRQAPGQGLEWMGRISPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYS YDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNV GDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFG QGTKLEIK SEQ ID NO: 157 VLDIQMTQSPSFLSASVGDRVTITCKASQNVGDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 158 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYI HWVRQAPGQGLEWMGRISPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYS YDVLDYWGQGTTVTVSS A-H.29SEQ ID NO: 159 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGHDFHLWYIHWVRQAPGQGLEWMGRISPGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVGDRVAWHQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 160 VL DIQMTQSPSFLSASVGDRVTITCKASQNVGDRVAWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 161 VH QVQLVQSGAEVKKPGSSVKVSCKASGHDFHLWYIHWVRQAPGQGLEWMGRISPGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.31 SEQ ID NO: 162 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGHDFKLTYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 163 VLDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 164 VHQVQLVQSGAEVKKPGSSVKVSCKASGHDFKLTYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.31SEQ ID NO: 165 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGTDFHLWYIHWVRQAPGQGLEWMGRVFAGSGSYRYNEKFK GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPS RFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 166 VL DIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 167 VH QVQLVQSGAEVKKPGSSVKVSCKASGTDFHLWYIHWVRQAPGQGLEWMGRVFAGSGSYRYNEKFK GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.32 SEQ ID NO: 168 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRISAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VADRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 169 VLDIQMTQSPSFLSASVGDRVTITCKASQNVADRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 170 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRISAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.33SEQ ID NO: 171 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRISAGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVEDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRF SGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 172 VL DIQMTQSPSFLSASVGDRVTITCKASQNVEDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 173 VH QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYIHWVRQAPGQGLEWMGRISAGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.34 SEQ ID NO: 174 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTYI HWVRQAPGQGLEWMGRISPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYS YDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNV GNRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFG QGTKLEIK SEQ ID NO: 175 VLDIQMTQSPSFLSASVGDRVTITCKASQNVGNRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 176 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTYI HWVRQAPGQGLEWMGRISPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYS YDVLDYWGQGTTVTVSS A-H.35SEQ ID NO: 177 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKTYIHWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVEDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRF SGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 178 VL DIQMTQSPSFLSASVGDRVTITCKASQNVEDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 179 VH QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKTYIHWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.36 SEQ ID NO: 180 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGHDFKLTYI HWVRQAPGQGLEWMGRVSPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VEDRVAWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTF GQGTKLEIK SEQ ID NO: 181 VLDIQMTQSPSFLSASVGDRVTITCKASQNVEDRVA WHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 182 VHQVQLVQSGAEVKKPGSSVKVSCKASGHDFKLTYI HWVRQAPGQGLEWMGRVSPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.37SEQ ID NO: 183 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKTYIHWVRQAPGQGLEWMGRIYPGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVADRVAWYQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 184 VL DIQMTQSPSFLSASVGDRVTITCKASQNVADRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 185 VH QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKTYIHWVRQAPGQGLEWMGRIYPGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.38 SEQ ID NO: 186 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKTYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 187 VLDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 188 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKTYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.39SEQ ID NO: 189 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYIHWVRQAPGQGLEWMGRISAGSGNIKYNEKFKGR VTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGS GGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFS GSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 190 VL DIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 191 VH QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYIHWVRQAPGQGLEWMGRISAGSGNIKYNEKFKGR VTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.40 SEQ ID NO: 192 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VGDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 193 VLDIQMTQSPSFLSASVGDRVTITCKASQNVGDRVA WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 194 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIYI HWVRQAPGQGLEWMGRISAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.41SEQ ID NO: 195 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGGTFKLTYIHWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 196 VL DIQMTQSPSFLSASVGDRVTITCKASQNVDDRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 197 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFKLTYIHWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.42 SEQ ID NO: 198 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYI HWVRQAPGQGLEWMGRISPGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VDNRVAWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 199 VLDIQMTQSPSFLSASVGDRVTITCKASQNVDNRVA WHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKL EIK SEQ ID NO: 200 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTYI HWVRQAPGQGLEWMGRISPGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.43SEQ ID NO: 201 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKFYIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVDNRVAWYQQKPGKAPKALIYSSSHRYKGVPSR FSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 202 VL DIQMTQSPSFLSASVGDRVTITCKASQNVDNRVAWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 203 VH QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKFYIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.44 SEQ ID NO: 204 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKFYI HWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VGDRVVWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLT FGQGTKLEIK SEQ ID NO: 205 VHQVQLVQSGAEVKKPGSSVKVSCKASGTDFDKFYI HWVRQAPGQGLEWMGRVSAGSGNVKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.45SEQ ID NO: 206 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFSAGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAVSYYSYDVLDYWGQGITVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSNITSASVGDRVTITCKASQNVGINVVWHQQKPGKAPKALIYSSSHRYSGVPSRF SGSGSGTEFTLTISSOPEDFAIATCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 207 VH QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFSAGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAVSYYSYDVLDYWGQGTTVTVSS A-H.46 SEQ ID NO: 208 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGYSFITYYI HWVRQAPGQGLEWMGWFSAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYAVGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VGINVVWFIQQKPGKAPKALIISSSIIRYSGVPSRFSGSGSGTFTTLTISSLQPEDFATYFCQQFKSYPLTF GQGTKLEIK SEQ ID NO: 209 VHQVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYI HWVRQAPGQGLEWMGWFSAGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.47SEQ ID NO: 210 VH + VL QVQLVQSGAEVIKKPGSSVIKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQMTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSELSASVGDRVTITCKASQNVGINVATWIIQQKPGKAPKALIYSSSHRYSGVPSRF SGSGSGIEFTLTISSLQPEDFATYKQQFKSYPLTFGQGTKLEIK SEQ ID NO: 211 VH QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.48 SEQ ID NO: 212 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYI IMIVRQAPGQGLEWMGWFSPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAVSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VGINVVWHQQKPGKAPKALIYSSSHRYSGVPSRFSGSGSGTEFILTISSOPEDFATYFCQQFKSYPLTF GQGTKLEIK SEQ ID NO: 213 VHQVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYI HWVRQAPGQGLEWMGWFSPGSGNTKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAVSY YSYDVLDYWGQGTTVTVSS A-H.49SEQ ID NO: 214 VH + VL QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLENVMGWFSPGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVGINVVWHQQKPGKAPKALIYSSSHRYSGVPSRF SGSGSGTETTLITSSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 215 VH QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFSPGSGNTKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.50 SEQ ID NO: 216 VH + VLQVQLVNQSGAEVKKPGSSVKVSCKASGYSFTTYYI HWVRQAPGQGLEWMGRIFPGSGINIKYNEKEKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYS YDVLDYWGQGTIVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNV GINVVWHQQKPGKAPKALIYSSSHRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFG QGTKLEIK SEQ ID NO: 217 VHQVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYI HWVRQAPGQGLEWMGRIFPGSGNIKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYS YDVLDYWGQGTTVTVSS A-H.51SEQ ID NO: 218 VH + VL QVQLVQSGAEVKKPGSSVENSCKASGYSFITYYIHWVRQAPGQGLEWMGINTFPGSGNIKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVCAGSIYSAGVLDYVIGQGTINTVSSGGGGSGGGGSGGGGS GGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVGINVVWHQQKPGKAPKALAYSSSHRYSGVPSRFS GSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 219 VH QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSIYSAGVLDYWGQGTTVTVSS A-H.52 SEQ ID NO: 220 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGYSFTLGYI HWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VGINVVWHQQKPGKAPKALIYSSSHRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTF GQGTKLEIK SEQ ID NO: 221 VHQVQLVQSGAEVKKPGSSVKVSCKASGYSFTLGYI HWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS A-H.53SEQ ID NO: 222 VH+ 30VL QVQLVQSGAEVKKPGSSVKVSCKASGYSFRLTYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVGINVVWHQQKPGKAPKALIYSSSHRYSGVPSRF SGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIK SEQ ID NO: 223 VH QVQLVQSGAEVKKPGSSVKVSCKASGYSFRLTYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKG RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSS A-H.54 SEQ ID NO: 224 VH + VLQVQLVQSGAEVKKPGSSVKVSCKASGYSFHNWY IHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQN VGINVVWHQQKPGKAPKALIYSSSHRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTF GQGTKLEIK SEQ ID NO: 225 VHQVQLVQSGAEVKKPGSSVKVSCKASGYSFHNWY IHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY YSYDVLDYWGQGTTVTVSS

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a VH and/or aVL of an antibody described in Table 1, or a sequence with at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a VH and a VLof an antibody described in Table 1, or a sequence with at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, an anti-TCRβV antibody disclosed herein has anantigen binding domain having a VL having a consensus sequence of SEQ IDNO: 230, wherein position 30 is G, E, A or D; position 31 is N or D;position 32 is R or K; position 36 is Y or H; and/or position 56 is K orS.

In some embodiments, an anti-TCRβV antibody disclosed herein has anantigen binding domain having a VH having a consensus sequence of SEQ IDNO: 231, wherein: position 27 is H or T or G or Y; position 28 is D or Tor S; position 30 is H or R or D or K or T; position 31 is L or D or Kor T or N; position 32 is W or F or T or I or Y or G; position 49 is Ror W; position 50 is V or I or F; position 51 is F or S or Y; position52 is A or P; position 56 is N or S; position 57 is T or V or Y or I;position 58 is K or R; position 97 is G or V; position 99 is Y or I;position 102 is Y or A; and/or position 103 is D or G.

Anti-TCRβ V12 Antibodies

Accordingly, in one aspect, the disclosure provides an anti-TCRβVantibody molecule that binds to human TCRβ V12, e.g., a TCRβ V12subfamily comprising: TCRβ V12-4*01, TCRβ V12-3*01 or TCRβ V12-5*01. Insome embodiments the TCRβ V12 subfamily comprises TCRβ V12-4*01. In someembodiments the TCRβ V12 subfamily comprises TCRβ V12-3*01.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, is a non-murine antibody molecule, e.g., a humanor humanized antibody molecule. In some embodiments, the anti-TCRβVantibody molecule, e.g., anti-TCRβ V12 antibody molecule is a humanantibody molecule. In some embodiments, the anti-TCRβV antibodymolecule, e.g., anti-TCRβ V12 antibody molecule is a humanized antibodymolecule.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, is isolated or recombinant.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, comprises at least one antigen-binding region,e.g., a variable region or an antigen-binding fragment thereof, from anantibody described herein, e.g., an antibody described in Table 2, orencoded by a nucleotide sequence in Table 2, or a sequence substantiallyidentical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% orhigher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, comprises at least one, two, three or fourvariable regions from an antibody described herein, e.g., an antibody asdescribed in Table 2, or encoded by a nucleotide sequence in Table 2, ora sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, comprises at least one or two heavy chainvariable regions from an antibody described herein, e.g., an antibody asdescribed in Table 2, or encoded by a nucleotide sequence in Table 2, ora sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, comprises at least one or two light chainvariable regions from an antibody described herein, e.g., an antibody asdescribed in Table 2, or encoded by a nucleotide sequence in Table 2, ora sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, comprises a heavy chain constant region for anIgG4, e.g., a human IgG4. In still another embodiment, the anti-TCRβVantibody molecule, e.g., anti-TCRβ V12 antibody molecule, includes aheavy chain constant region for an IgG1, e.g., a human IgG1. In oneembodiment, the heavy chain constant region comprises an amino sequenceset forth in Table 3, or a sequence substantially identical (e.g., atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical)thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, includes a kappa light chain constant region,e.g., a human kappa light chain constant region. In one embodiment, thelight chain constant region comprises an amino sequence set forth inTable 3, or a sequence substantially identical (e.g., at least 80%, 85%,90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, includes at least one, two, or threecomplementarity determining regions (CDRs) from a heavy chain variableregion of an antibody described herein, e.g., an antibody as describedin Table 2, or encoded by the nucleotide sequence in Table 2, or asequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, includes at least one, two, or three CDRs (orcollectively all of the CDRs) from a heavy chain variable regioncomprising an amino acid sequence shown in Table 2, or encoded by anucleotide sequence shown in Table 2. In one embodiment, one or more ofthe CDRs (or collectively all of the CDRs) have one, two, three, four,five, six or more changes, e.g., amino acid substitutions or deletions,relative to the amino acid sequence shown in Table 2, or encoded by anucleotide sequence shown in Table 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, includes at least one, two, or threecomplementarity determining regions (CDRs) from a light chain variableregion of an antibody described herein, e.g., an antibody as describedin Table 2, or encoded by the nucleotide sequence in Table 2, or asequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, includes at least one, two, or three CDRs (orcollectively all of the CDRs) from a light chain variable regioncomprising an amino acid sequence shown in Table 2, or encoded by anucleotide sequence shown in Table 2. In one embodiment, one or more ofthe CDRs (or collectively all of the CDRs) have one, two, three, four,five, six or more changes, e.g., amino acid substitutions or deletions,relative to the amino acid sequence shown in Table 2, or encoded by anucleotide sequence shown in Table 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, includes at least one, two, three, four, five orsix CDRs (or collectively all of the CDRs) from a heavy and light chainvariable region comprising an amino acid sequence shown in Table 2, orencoded by a nucleotide sequence shown in Table 2. In one embodiment,one or more of the CDRs (or collectively all of the CDRs) have one, two,three, four, five, six or more changes, e.g., amino acid substitutionsor deletions, relative to the amino acid sequence shown in Table 2, orencoded by a nucleotide sequence shown in Table 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, molecule includes all six CDRs from an antibodydescribed herein, e.g., an antibody as described in Table 2, or encodedby the nucleotide sequence in Table 2, or closely related CDRs, e.g.,CDRs which are identical or which have at least one amino acidalteration, but not more than two, three or four alterations (e.g.,substitutions, deletions, or insertions, e.g., conservativesubstitutions). In some embodiments, the anti-TCRβV antibody molecule,e.g., anti-TCRβ V12 antibody molecule, may include any CDR describedherein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes at least one, two, or three CDRsaccording to Kabat et al. (e.g., at least one, two, or three CDRsaccording to the Kabat definition as set out in Table 2) from a heavychain variable region of an antibody described herein, e.g., an antibodychosen as described in Table 2, or a sequence substantially identical(e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higheridentical) to any of the aforesaid sequences; or which have at least oneamino acid alteration, but not more than two, three or four alterations(e.g., substitutions, deletions, or insertions, e.g., conservativesubstitutions) relative to one, two, or three CDRs according to Kabat etal. shown in Table 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes at least one, two, or three CDRsaccording to Kabat et al. (e.g., at least one, two, or three CDRsaccording to the Kabat definition as set out in Table 2) from a lightchain variable region of an antibody described herein, e.g., an antibodyas described in Table 2, or a sequence substantially identical (e.g., atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to anyof the aforesaid sequences; or which have at least one amino acidalteration, but not more than two, three or four alterations (e.g.,substitutions, deletions, or insertions, e.g., conservativesubstitutions) relative to one, two, or three CDRs according to Kabat etal. shown in Table 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes at least one, two, three, four, five, orsix CDRs according to Kabat et al. (e.g., at least one, two, three,four, five, or six CDRs according to the Kabat definition as set out inTable 2) from the heavy and light chain variable regions of an antibodydescribed herein, e.g., an antibody as described in Table 2, or encodedby the nucleotide sequence in Table 2; or a sequence substantiallyidentical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% orhigher identical) to any of the aforesaid sequences; or which have atleast one amino acid alteration, but not more than two, three or fouralterations (e.g., substitutions, deletions, or insertions, e.g.,conservative substitutions) relative to one, two, three, four, five, orsix CDRs according to Kabat et al. shown in Table 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes all six CDRs according to Kabat et al.(e.g., all six CDRs according to the Kabat definition as set out inTable 2) from the heavy and light chain variable regions of an antibodydescribed herein, e.g., an antibody as described in Table 2, or encodedby the nucleotide sequence in Table 2; or encoded by the nucleotidesequence in Table 2; or a sequence substantially identical (e.g., atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to anyof the aforesaid sequences; or which have at least one amino acidalteration, but not more than two, three or four alterations (e.g.,substitutions, deletions, or insertions, e.g., conservativesubstitutions) relative to all six CDRs according to Kabat et al. shownin Table 2. In some embodiments, the anti-TCRβV antibody molecule, e.g.,anti-TCRβ V12 antibody molecule may include any CDR described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes at least one, two, or three hypervariableloops that have the same canonical structures as the correspondinghypervariable loop of an antibody described herein, e.g., an antibodydescribed in Table 2, e.g., the same canonical structures as at leastloop 1 and/or loop 2 of the heavy and/or light chain variable domains ofan antibody described herein. See, e.g., Chothia et al., (1992) J. Mol.Biol. 227:799-817; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798for descriptions of hypervariable loop canonical structures. Thesestructures can be determined by inspection of the tables described inthese references.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes at least one, two, or three CDRsaccording to Chothia et al. (e.g., at least one, two, or three CDRsaccording to the Chothia definition as set out in Table 2) from a heavychain variable region of an antibody described herein, e.g., an antibodychosen as described in Table 2, or a sequence substantially identical(e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higheridentical) to any of the aforesaid sequences; or which have at least oneamino acid alteration, but not more than two, three or four alterations(e.g., substitutions, deletions, or insertions, e.g., conservativesubstitutions) relative to one, two, or three CDRs according to Chothiaet al. shown in Table 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes at least one, two, or three CDRsaccording to Chothia et al. (e.g., at least one, two, or three CDRsaccording to the Chothia definition as set out in Table 2) from a lightchain variable region of an antibody described herein, e.g., an antibodyas described in Table 2, or a sequence substantially identical (e.g., atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to anyof the aforesaid sequences; or which have at least one amino acidalteration, but not more than two, three or four alterations (e.g.,substitutions, deletions, or insertions, e.g., conservativesubstitutions) relative to one, two, or three CDRs according to Chothiaet al. shown in Table 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes at least one, two, three, four, five, orsix CDRs according to Chothia et al. (e.g., at least one, two, three,four, five, or six CDRs according to the Chothia definition as set outin Table 2) from the heavy and light chain variable regions of anantibody described herein, e.g., an antibody as described in Table 2, orencoded by the nucleotide sequence in Table 2; or a sequencesubstantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or higher identical) to any of the aforesaid sequences; orwhich have at least one amino acid alteration, but not more than two,three or four alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions) relative to one, two,three, four, five, or six CDRs according to Chothia et al. shown inTable 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes all six CDRs according to Chothia et al.(e.g., all six CDRs according to the Chothia definition as set out inTable 2) from the heavy and light chain variable regions of an antibodydescribed herein, e.g., an antibody as described in Table 2, or encodedby the nucleotide sequence in Table 2; or encoded by the nucleotidesequence in Table 2; or a sequence substantially identical (e.g., atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to anyof the aforesaid sequences; or which have at least one amino acidalteration, but not more than two, three or four alterations (e.g.,substitutions, deletions, or insertions, e.g., conservativesubstitutions) relative to all six CDRs according to Chothia et al.shown in Table 2. In some embodiments, the anti-TCRβV antibody molecule,e.g., anti-TCRβ V12 antibody molecule may include any CDR describedherein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes at least one, two, or three CDRsaccording to a combined CDR (e.g., at least one, two, or three CDRsaccording to the combined CDR definition as set out in Table 2) from aheavy chain variable region of an antibody described herein, e.g., anantibody chosen as described in Table 2, or a sequence substantiallyidentical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% orhigher identical) to any of the aforesaid sequences; or which have atleast one amino acid alteration, but not more than two, three or fouralterations (e.g., substitutions, deletions, or insertions, e.g.,conservative substitutions) relative to one, two, or three CDRsaccording to combined CDR shown in Table 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes at least one, two, or three CDRsaccording to a combined CDR (e.g., at least one, two, or three CDRsaccording to the combined CDR definition as set out in Table 2) from alight chain variable region of an antibody described herein, e.g., anantibody as described in Table 2, or a sequence substantially identical(e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higheridentical) to any of the aforesaid sequences; or which have at least oneamino acid alteration, but not more than two, three or four alterations(e.g., substitutions, deletions, or insertions, e.g., conservativesubstitutions) relative to one, two, or three CDRs according to acombined CDR shown in Table 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes at least one, two, three, four, five, orsix CDRs according to a combined CDR. (e.g., at least one, two, three,four, five, or six CDRs according to the combined CDR definition as setout in Table 2) from the heavy and light chain variable regions of anantibody described herein, e.g., an antibody as described in Table 2, orencoded by the nucleotide sequence in Table 2; or a sequencesubstantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or higher identical) to any of the aforesaid sequences; orwhich have at least one amino acid alteration, but not more than two,three or four alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions) relative to one, two,three, four, five, or six CDRs according to a combined CDR shown inTable 2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes all six CDRs according to a combined CDR(e.g., all six CDRs according to the combined CDR definition as set outin Table 2) from the heavy and light chain variable regions of anantibody described herein, e.g., an antibody as described in Table 2, orencoded by the nucleotide sequence in Table 2; or encoded by thenucleotide sequence in Table 2; or a sequence substantially identical(e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higheridentical) to any of the aforesaid sequences; or which have at least oneamino acid alteration, but not more than two, three or four alterations(e.g., substitutions, deletions, or insertions, e.g., conservativesubstitutions) relative to all six CDRs according to a combined CDRshown in Table 2. In some embodiments, the anti-TCRβV antibody molecule,e.g., anti-TCRβ V12 antibody molecule may include any CDR describedherein.

In some embodiments, a combined CDR as set out in Table 1 is a CDR thatcomprises a Kabat CDR and a Chothia CDR.

In some embodiments, the anti-TCRβV antibody molecule, e e.g., anti-TCRβV12 antibody molecule, molecule includes a combination of CDRs orhypervariable loops identified as combined CDRs in Table 1. In someembodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12antibody molecule, can contain any combination of CDRs or hypervariableloops according the “combined” CDRs are described in Table 1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes a combination of CDRs or hypervariableloops defined according to the Kabat et al. and Chothia et al., or asdescribed in Table 1

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule can contain any combination of CDRs orhypervariable loops according to the Kabat and Chothia definitions.

In an embodiment, e.g., an embodiment comprising a variable region, aCDR (e.g., a combined CDR, Chothia CDR or Kabat CDR), or other sequencereferred to herein, e.g., in Table 2, the antibody molecule is amonospecific antibody molecule, a bispecific antibody molecule, abivalent antibody molecule, a biparatopic antibody molecule, or anantibody molecule that comprises an antigen binding fragment of anantibody, e.g., a half antibody or antigen binding fragment of a halfantibody. In certain embodiments the antibody molecule comprises amultispecific molecule, e.g., a bispecific molecule, e.g., as describedherein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes:

(i) one, two or all of a light chain complementarity determining region1 (LC CDR1), a light chain complementarity determining region 2 (LCCDR2), and a light chain complementarity determining region 3 (LC CDR3)of SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ IDNO: 29 or SEQ ID NO: 30, and/or(ii) one, two or all of a heavy chain complementarity determining region1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2),and a heavy chain complementarity determining region 3 (HC CDR3) of SEQID NO: 15, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

(i) a LC CDR1 amino acid sequence of SEQ ID NO: 20, a LC CDR2 amino acidsequence of SEQ ID NO: 21, or a LC CDR3 amino acid sequence of SEQ IDNO: 22; and/or(ii) a HC CDR1 amino acid sequence of SEQ ID NO: 17, a HC CDR2 aminoacid sequence of SEQ ID NO: 18, or a HC CDR3 amino acid sequence of SEQID NO: 19.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

(i) a light chain variable region (VL) comprising a LC CDR1 amino acidsequence of SEQ ID NO: 20, a LC CDR2 amino acid sequence of SEQ ID NO:21, and a LC CDR3 amino acid sequence of SEQ ID NO: 2; and/or(ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acidsequence of SEQ ID NO: 17, a HC CDR2 amino acid sequence of SEQ ID NO:18, and a HC CDR3 amino acid sequence of SEQ ID NO: 19.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

(i) a LC CDR1 amino acid sequence of SEQ ID NO: 63, a LC CDR2 amino acidsequence of SEQ ID NO: 64, or a LC CDR3 amino acid sequence of SEQ IDNO: 65; and/or(ii) a HC CDR1 amino acid sequence of SEQ ID NO: 57, a HC CDR2 aminoacid sequence of SEQ ID NO: 58, or a HC CDR3 amino acid sequence of SEQID NO: 59.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

(i) a light chain variable region (VL) comprising a LC CDR1 amino acidsequence of SEQ ID NO: 63, a LC CDR2 amino acid sequence of SEQ ID NO:64, or a LC CDR3 amino acid sequence of SEQ ID NO: 65; and/or(ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acidsequence of SEQ ID NO: 57, a HC CDR2 amino acid sequence of SEQ ID NO:58, or a HC CDR3 amino acid sequence of SEQ ID NO: 59.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

(i) a LC CDR1 amino acid sequence of SEQ ID NO: 66, a LC CDR2 amino acidsequence of SEQ ID NO: 67, or a LC CDR3 amino acid sequence of SEQ IDNO: 68; and/or(ii) a HC CDR1 amino acid sequence of SEQ ID NO: 60, a HC CDR2 aminoacid sequence of SEQ ID NO: 61, or a HC CDR3 amino acid sequence of SEQID NO: 62.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

(i) a light chain variable region (VL) comprising a LC CDR1 amino acidsequence of SEQ ID NO: 63, a LC CDR2 amino acid sequence of SEQ ID NO:64, or a LC CDR3 amino acid sequence of SEQ ID NO: 65; and/or(ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acidsequence of SEQ ID NO: 57, a HC CDR2 amino acid sequence of SEQ ID NO:58, or a HC CDR3 amino acid sequence of SEQ ID NO: 59.

In one embodiment, the light or the heavy chain variable framework(e.g., the region encompassing at least FR1, FR2, FR3, and optionallyFR4) of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibodymolecule can be chosen from: (a) a light or heavy chain variableframework including at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%,or 100% of the amino acid residues from a human light or heavy chainvariable framework, e.g., a light or heavy chain variable frameworkresidue from a human mature antibody, a human germline sequence, or ahuman consensus sequence; (b) a light or heavy chain variable frameworkincluding from 20% to 80%, 40% to 60%, 60% to 90%, or 70% to 95% of theamino acid residues from a human light or heavy chain variableframework, e.g., a light or heavy chain variable framework residue froma human mature antibody, a human germline sequence, or a human consensussequence; (c) a non-human framework (e.g., a rodent framework); or (d) anon-human framework that has been modified, e.g., to remove antigenic orcytotoxic determinants, e.g., deimmunized, or partially humanized. Inone embodiment, the light or heavy chain variable framework region(particularly FR1, FR2 and/or FR3) includes a light or heavy chainvariable framework sequence at least 70, 75, 80, 85, 87, 88, 90, 92, 94,95, 96, 97, 98, 99% identical or identical to the frameworks of a VL orVH segment of a human germline gene.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule, comprises a heavy chain variable domain having atleast one, two, three, four, five, six, seven, ten, fifteen, twenty ormore changes, e.g., amino acid substitutions or deletions, from an aminoacid sequence described in Table 2. e.g., the amino acid sequence of theFR region in the entire variable region, e.g., shown in FIGS. 2A and 2B,or in SEQ ID NOs: 23-25.

Alternatively, or in combination with the heavy chain substitutionsdescribed herein the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12antibody molecule comprises a light chain variable domain having atleast one, two, three, four, five, six, seven, ten, fifteen, twenty ormore amino acid changes, e.g., amino acid substitutions or deletions,from an amino acid sequence of an antibody described herein e.g., theamino acid sequence of the FR region in the entire variable region,e.g., shown in FIGS. 2A and 2B, or in SEQ ID NOs: 26-30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes one, two, three, or four heavy chainframework regions shown in FIG. 2A, or a sequence substantiallyidentical thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule includes one, two, three, or four light chainframework regions shown in FIG. 2B, or a sequence substantiallyidentical thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises the light chain framework region 1 e.g.,as shown in FIG. 2B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises the light chain framework region 2 e.g.,as shown in FIG. 2B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises the light chain framework region 3,e.g., as shown in FIG. 2B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises the light chain framework region 4,e.g., as shown in FIG. 2B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises a light chain comprising a frameworkregion, e.g., framework region 1 (FR1), comprising a change, e.g., asubstitution (e.g., a conservative substitution) at one or more, e.g.,all, position disclosed herein according to Kabat numbering. In someembodiments, FR1 comprises an Aspartic Acid at position 1, e.g., asubstitution at position 1 according to Kabat numbering, e.g., anAlanine to Aspartic Acid substitution. In some embodiments, FR1comprises an Asparagine at position 2, e.g., a substitution at position2 according to Kabat numbering, e.g., an Isoleucine to Asparaginesubstitution, Serine to Asparagine substitution or Tyrosine toAsparagine substitution. In some embodiments, FR1 comprises a Leucine atposition 4, e.g., a substitution at position 4 according to Kabatnumbering, e.g., a Methionine to Leucine substitution.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises a light chain comprising a frameworkregion, e.g., framework region 1 (FR1), comprising a substitution atposition 1 according to Kabat numbering, e.g., an Alanine to AsparticAcid substitution, a substitution at position 2 according to Kabatnumbering, e.g., an Isoleucine to Asparagine substitution, Serine toAsparagine substitution or Tyrosine to Asparagine substitution, and asubstitution at position 4 according to Kabat numbering, e.g., aMethionine to Leucine substitution. In some embodiments, the anti-TCRβVantibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises alight chain comprising a framework region, e.g., framework region 1(FR1), comprising a substitution at position 1 according to Kabatnumbering, e.g., an Alanine to Aspartic Acid substitution, and asubstitution at position 2 according to Kabat numbering, e.g., anIsoleucine to Asparagine substitution, Serine to Asparagine substitutionor Tyrosine to Asparagine substitution. In some embodiments, theanti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody moleculecomprises a light chain comprising a framework region, e.g., frameworkregion 1 (FR1), comprising a substitution at position 1 according toKabat numbering, e.g., an Alanine to Aspartic Acid substitution, and asubstitution at position 4 according to Kabat numbering, e.g., aMethionine to Leucine substitution. In some embodiments, the anti-TCRβVantibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises alight chain comprising a framework region, e.g., framework region 1(FR1), comprising a substitution at position 2 according to Kabatnumbering, e.g., an Isoleucine to Asparagine substitution, Serine toAsparagine substitution or Tyrosine to Asparagine substitution, and asubstitution at position 4 according to Kabat numbering, e.g., aMethionine to Leucine substitution. In some embodiments, thesubstitution is relative to a human germline light chain frameworkregion sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises a light chain comprising a frameworkregion, e.g., framework region 3 (FR3), comprising a change, e.g., asubstitution (e.g., a conservative substitution) at one or more, e.g.,all, position disclosed herein according to Kabat numbering. In someembodiments, FR3 comprises a Glycine at position 66, e.g., asubstitution at position 66 according to Kabat numbering, e.g., a Lysineto Glycine substitution, or a Serine to Glycine substitution. In someembodiments, FR3 comprises an Asparagine at position 69, e.g., asubstitution at position 69 according to Kabat numbering, e.g., aTyrosine to Asparagine substitution. In some embodiments, FR3 comprisesa Tyrosine at position 71, e.g., a substitution at position 71 accordingto Kabat numbering, e.g., a Phenylalanine to Tyrosine substitution, oran Alanine to Tyrosine substitution.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises a light chain comprising a frameworkregion, e.g., framework region 3 (FR3), comprising a substitution atposition 66 according to Kabat numbering, e.g., a Lysine to Glycinesubstitution, or a Serine to Glycine substitution, and a substitution atposition 69 according to Kabat numbering, e.g., a Tyrosine to Asparaginesubstitution. In some embodiments, the anti-TCRβV antibody molecule,e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprisinga framework region, e.g., framework region 3 (FR3), comprising asubstitution at position 66 according to Kabat numbering, e.g., Lysineto Glycine substitution, or a Serine to Glycine substitution, and asubstitution at position 71 according to Kabat numbering, e.g., aPhenylalanine to Tyrosine substitution, or an Alanine to Tyrosinesubstitution. In some embodiments, the anti-TCRβV antibody molecule,e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprisinga framework region, e.g., framework region 3 (FR3), comprising asubstitution at position 69 according to Kabat numbering, e.g., aTyrosine to Asparagine substitution and a substitution at position 71according to Kabat numbering, e.g., a Phenylalanine to Tyrosinesubstitution, or an Alanine to Tyrosine substitution. In someembodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12antibody molecule comprises a light chain comprising a framework region,e.g., framework region 3 (FR3), comprising a substitution at position 66according to Kabat numbering, e.g., a Lysine to Glycine substitution, ora Serine to Glycine substitution, a substitution at position 69according to Kabat numbering, e.g., a Tyrosine to Asparaginesubstitution and a substitution at position 71 according to Kabatnumbering, e.g., a Phenylalanine to Tyrosine substitution, or an Alanineto Tyrosine substitution. In some embodiments, the substitution isrelative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises a light chain comprising: a frameworkregion 1 (FR1) comprising a substitution at position 2 according toKabat numbering, e.g., a Isoleucine to Asparagine substitution; and aframework region 3 (FR3), comprising a substitution at position 69according to Kabat numbering, e.g., a Threonine to Asparaginesubstitution and a substitution at position 71 according to Kabatnumbering, e.g., a Phenylalanine to Tyrosine substitution, e.g., asshown in the amino acid sequence of SEQ ID NO: 26. In some embodiments,the substitution is relative to a human germline light chain frameworkregion sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises a light chain comprising: (a) aframework region 1 (FR1) comprising a substitution at position 1according to Kabat numbering, e.g., a Alanine to Aspartic Acidsubstitution, and a substitution at position 2 according to Kabatnumbering, e.g., a Isoleucine to Asparagine substitution; and (b) aframework region 3 (FR3), comprising a substitution at position 69according to Kabat numbering, e.g., a Threonine to Asparaginesubstitution and a substitution at position 71 according to Kabatnumbering, e.g., a Phenylalanine to Tyrosine substitution, e.g., asshown in the amino acid sequence of SEQ ID NO: 27 In some embodiments,the substitution is relative to a human germline light chain frameworkregion sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises a light chain comprising: (a) aframework region 1 (FR1) comprising a substitution at position 2according to Kabat numbering, e.g., a Serine to Asparagine substitution;and a substitution at position 4 according to Kabat numbering, e.g., aMethionine to Leucine substitution; and (b) a framework region 3 (FR3),comprising a substitution at position 69 according to Kabat numbering,e.g., a Threonine to Asparagine substitution and a substitution atposition 71 according to Kabat numbering, e.g., a Phenylalanine toTyrosine substitution, e.g., as shown in the amino acid sequence of SEQID NO: 28 In some embodiments, the substitution is relative to a humangermline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises a light chain comprising: (a) aframework region 1 (FR1) comprising a substitution at position 2according to Kabat numbering, e.g., a Serine to Asparagine substitution;and (b) a framework region 3 (FR3) comprising a substitution at position66 according to Kabat numbering, e.g., a Lysine to Glycine substitution;a substitution at position 69 according to Kabat numbering, e.g., aThreonine to Asparagine substitution; and a substitution at position 71according to Kabat numbering, e.g., a Alanine to Tyrosine substitution,e.g., as shown in the amino acid sequence of SEQ ID NO: 29. In someembodiments, the substitution is relative to a human germline lightchain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises a light chain comprising: (a) aframework region 1 (FR1) comprising a substitution at position 2according to Kabat numbering, e.g., a Tyrosine to Asparaginesubstitution; and (b) a framework region 3 (FR3) comprising asubstitution at position 66 according to Kabat numbering, e.g., a Serineto Glycine substitution; a substitution at position 69 according toKabat numbering, e.g., a Threonine to Asparagine substitution; and asubstitution at position 71 according to Kabat numbering, e.g., aAlanine to Tyrosine substitution, e.g., as shown in the amino acidsequence of SEQ ID NO: 29. In some embodiments, the substitution isrelative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises a light chain variable domaincomprising: (a) a framework region 1 (FR1) comprising a change, e.g., asubstitution (e.g., a conservative substitution) at one or more (e.g.,all) positions disclosed herein according to Kabat numbering, and (b) aframework region 3 (FR3) comprising a change, e.g., a substitution(e.g., a conservative substitution) at one or more (e.g., all) positiondisclosed herein according to Kabat numbering. In some embodiments, thesubstitution is relative to a human germline light chain frameworkregion sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises the heavy chain framework region 1,e.g., as shown in FIG. 2A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises the heavy chain framework region 2,e.g., as shown in FIG. 2A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises the heavy chain framework region 3,e.g., as shown in FIG. 2A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises the heavy chain framework region 4,e.g., as shown in FIG. 2A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises the heavy chain framework regions 1-4,e.g., SEQ ID NOS: 20-23, or as shown in FIG. 2A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises the light chain framework regions 1-4,e.g., SEQ ID NOs: 26-30, or as shown in FIG. 2B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises the heavy chain framework regions 1-4,e.g., SEQ ID NOs: 23-25; and the light chain framework regions 1-4,e.g., SEQ ID NOs: 26-30, or as shown in FIGS. 2A and 2B.

In some embodiments, the heavy or light chain variable domain, or both,of, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibodymolecule includes an amino acid sequence, which is substantiallyidentical to an amino acid disclosed herein, e.g., at least 80%, 85%,90%, 92%, 95%, 97%, 98%, 99% or higher identical to a variable region ofan antibody described herein, e.g., an antibody as described in Table 2,or encoded by the nucleotide sequence in Table 2; or which differs atleast 1 or 5 residues, but less than 40, 30, 20, or 10 residues, from avariable region of an antibody described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises at least one, two, three, or fourantigen-binding regions, e.g., variable regions, having an amino acidsequence as set forth in Table 2, or a sequence substantially identicalthereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or moreidentical thereto, or which differs by no more than 1, 2, 5, 10, or 15amino acid residues from the sequences shown in Table 2. In anotherembodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12antibody molecule includes a VH and/or VL domain encoded by a nucleicacid having a nucleotide sequence as set forth in Table 2, or a sequencesubstantially identical thereto (e.g., a sequence at least about 85%,90%, 95%, 99% or more identical thereto, or which differs by no morethan 3, 6, 15, 30, or 45 nucleotides from the sequences shown in Table2.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising an amino acid sequence chosen from the amino acidsequence of SEQ ID NO: 23, SEQ ID NO:24 or SEQ ID NO:25, an amino acidsequence at least about 85%, 90%, 95%, 99% or more identical to theamino acid sequence SEQ ID NO: 23, SEQ ID NO:24 or SEQ ID NO:25, or anamino acid sequence which differs by no more than 1, 2, 5, 10, or 15amino acid residues from the amino acid sequence of SEQ ID NO: 23, SEQID NO:24 or SEQ ID NO:25; and/ora VL domain comprising an amino acid sequence chosen from the amino acidsequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29or SEQ ID NO: 30, an amino acid sequence at least about 85%, 90%, 95%,99% or more identical to the amino acid sequence of SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30, or an aminoacid sequence which differs by no more than 1, 2, 5, 10, or 15 aminoacid residues from the amino acid sequence of SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 23, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 23, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 23; and

a VL domain comprising the amino acid sequence of SEQ ID NO: 26, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 26, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 23, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 23, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 23; anda VL domain comprising the amino acid sequence of SEQ ID NO: 27, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 27, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 23, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 23, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 23; anda VL domain comprising the amino acid sequence of SEQ ID NO: 28, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 28, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 23, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 23, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 23; anda VL domain comprising the amino acid sequence of SEQ ID NO: 29, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 29, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 29.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 23, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 23, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 23; anda VL domain comprising the amino acid sequence of SEQ ID NO: 30, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 30, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 24, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 24, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 24; anda VL domain comprising the amino acid sequence of SEQ ID NO: 26, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 26, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 24, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 24, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 24; anda VL domain comprising the amino acid sequence of SEQ ID NO: 27, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 27, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 24, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 24, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 24; anda VL domain comprising the amino acid sequence of SEQ ID NO: 28, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 28, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 24, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 24, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 24; anda VL domain comprising the amino acid sequence of SEQ ID NO: 29, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 29, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 29.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 24, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 24, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 24; anda VL domain comprising the amino acid sequence of SEQ ID NO: 30, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 30, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 25, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 25, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 25; anda VL domain comprising the amino acid sequence of SEQ ID NO: 26, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 26, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 25, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 25, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 25; anda VL domain comprising the amino acid sequence of SEQ ID NO: 27, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 27, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 25, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 25, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 25; anda VL domain comprising the amino acid sequence of SEQ ID NO: 28, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 28, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 25, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 25, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 25; anda VL domain comprising the amino acid sequence of SEQ ID NO: 29, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 29, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 29.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule comprises:

a VH domain comprising the amino acid sequence of SEQ ID NO: 25, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 25, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 25; anda VL domain comprising the amino acid sequence of SEQ ID NO: 30, anamino acid sequence at least about 85%, 90%, 95%, 99% or more identicalto the amino acid sequence SEQ ID NO: 30, or an amino acid sequencewhich differs by no more than 1, 2, 5, 10, or 15 amino acid residuesfrom the amino acid sequence of SEQ ID NO: 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule is a full antibody or fragment thereof (e.g., aFab, F(ab′)2, Fv, or a single chain Fv fragment (scFv)). In embodiments,the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβV6-5*01) antibody molecule is a monoclonal antibody or an antibody withsingle specificity. In some embodiments, the anti-TCRβV antibodymolecule, e.g., anti-TCR3 V12 antibody molecule, can also be ahumanized, chimeric, camelid, shark, or an in vitro-generated antibodymolecule. In some embodiments, the anti-TCRβV antibody molecule, e.g.,anti-TCRβ V12 antibody molecule is a humanized antibody molecule. Theheavy and light chains of the anti-TCRβV antibody molecule, e.g.,anti-TCRβ V12 antibody molecule can be full-length (e.g., an antibodycan include at least one, and preferably two, complete heavy chains, andat least one, and preferably two, complete light chains) or can includean antigen-binding fragment (e.g., a Fab, F(ab′)2, Fv, a single chain Fvfragment, a single domain antibody, a diabody (dAb), a bivalentantibody, or bispecific antibody or fragment thereof, a single domainvariant thereof, or a camelid antibody).

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule is in the form of a multispecific molecule, e.g.,a bispecific molecule, e.g., as described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule has a heavy chain constant region (Fc) chosenfrom, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4,IgM, IgA1, IgA2, IgD, and IgE. In some embodiments, the Fc region ischosen from the heavy chain constant regions of IgG1, IgG2, IgG3, andIgG4. In some embodiments, the Fc region is chosen from the heavy chainconstant region of IgG1 or IgG2 (e.g., human IgG1, or IgG2). In someembodiments, the heavy chain constant region is human IgG1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV12 antibody molecule has a light chain constant region chosen from,e.g., the light chain constant regions of kappa or lambda, preferablykappa (e.g., human kappa). In one embodiment, the constant region isaltered, e.g., mutated, to modify the properties of the anti-TCRβVantibody molecule, e.g., anti-TCRβ V12 antibody molecule (e.g., toincrease or decrease one or more of: Fc receptor binding, antibodyglycosylation, the number of cysteine residues, effector cell function,or complement function). For example, the constant region is mutated atpositions 296 (M to Y), 298 (S to T), 300 (T to E), 477 (H to K) and 478(N to F) to alter Fc receptor binding (e.g., the mutated positionscorrespond to positions 132 (M to Y), 134 (S to T), 136 (T to E), 313 (Hto K) and 314 (N to F) of SEQ ID NOs: 212 or 214; or positions 135 (M toY), 137 (S to T), 139 (T to E), 316 (H to K) and 317 (N to F) of SEQ IDNOs: 215, 216, 217 or 218).

Antibody B-H.1 comprises a first chain comprising the amino acidsequence of SEQ ID NO: 3280 and a second chain comprising the amino acidsequence of SEQ ID NO: 3281.

TABLE 2 Amino acid and nucleotide sequences for murine and humanizedantibody molecules. The antibody molecules include murine mAbAntibody B and humanized mAb Antibody B-H.1. The amino acid theheavy and light chain CDRs, and the amino acid and nucleotidesequences of the heavy and light chain variable regions, andthe heavy and light chains are shown. Antibody B (murine) SEQ ID NO: 17HC CDR1 (Combined) GFTFSNFGMH SEQ ID NO: 18 HC CDR2 (Combined)YISSGSSTIYYADTLKG SEQ ID NO: 19 HC CDR3 (Combined) RGEGAMDYSEQ ID NO: 57 HC CDR1 (Kabat) NFGMH SEQ ID NO: 58 HC CDR2 (Kabat)YISSGSSTIYYADTLKG SEQ ID NO: 59 HC CDR3 (Kabat) RGEGAMDY SEQ ID NO: 60HC CDR1 (Chothia) GFTFSNF SEQ ID NO: 61 HC CDR2 (Chothia) SSGSSTSEQ ID NO: 62 HC CDR3 (Chothia) RGEGAMDY SEQ ID NO: 15 VHDVQLVESGGGLVQPGGSRKLSCAASGFTFSNFGM HWVRQAPDKGLEWVAYISSGSSTIYYADTLKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARRGEG AMDYWGQGTSVTVSS SEQ ID NO: 20LC CDR1 (Combined) RASSSVNYIY SEQ ID NO: 21 LC CDR2 (Combined)) YTSNLAPSEQ ID NO: 22 LC CDR3 (Combined) QQFTSSPFT SEQ ID NO: 63 LC CDR1 (Kabat)RASSSVNYIY SEQ ID NO: 64 LC CDR2 (Kabat) YTSNLAP SEQ ID NO: 65LC CDR3 (Kabat) QQFTSSPFT SEQ ID NO: 66 LC CDR1 (Chothia) RASSSVNYIYSEQ ID NO: 67 LC CDR2 (Chothia) YTSNLAP SEQ ID NO: 68 LC CDR3 (Chothia)QQFTSSPFT SEQ ID NO: 16 VL ENVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPTRFSGSGSGN SYSLTISSMEGEDAATYYCQQFTSSPFTFGSGTKLEIK Antibody B humanized (B-H) Antibody B-H.1A HC-1 SEQ ID NO: 17HC CDR1 (Combined) GFTFSNFGMH SEQ ID NO: 18 HC CDR2 (Combined)YISSGSSTIYYADTLKG SEQ ID NO: 19 HC CDR3 (Combined) RGEGAMDY SEQ ID NO:VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGM 3438HWVRQAPGKGLEWVSYISSGSSTIYYADTLKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARRGEGAMDYWGQGTTVTVSS SEQ ID NO: 31 DNA VH GAGGTGCAGCTGGTTGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGC CGCTTCTGGCTTCACCTTCTCCAACTTCGGCATGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGG AATGGGTGTCCTACATCTCCTCCGGCTCCTCCACCATCTACTACGCTGACACCCTGAAGGGCAGATTC ACCATCTCTCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACAC CGCCGTGTACTACTGTGCTAGAAGAGGCGAGGGCGCCATGGATTATTGGGGCCAGGGAACCACAGTGA CCGTGTCTAGC Antibody B-H.1B HC-2SEQ ID NO: 17 HC CDR1 (Combined) GFTFSNFGMH SEQ ID NO: 18HC CDR2 (Combined) YISSGSSTIYYADTLKG SEQ ID NO: 19 HC CDR3 (Combined)RGEGAMDY SEQ ID NO: 24 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVSYISSGSSTIYYADTLKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARRGEGAMDYWGQGTTVTVSS SEQ ID NO: 32 DNA VH GAGGTGCAGCTGGTTGAATCTGGCGGAGGATTGGTTCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGC CGCTTCTGGCTTCACCTTCTCCAACTTCGGCATGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGG AATGGGTGTCCTACATCTCCTCCGGCTCCTCCACCATCTACTACGCTGACACCCTGAAGGGCAGATTC ACCATCAGCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACAC CGCCGTGTACTACTGTGCTAGAAGAGGCGAGGGCGCCATGGATTATTGGGGCCAGGGAACCACAGTGA CCGTGTCTAGC Antibody B-H.1C HC-3SEQ ID NO: 17 HC CDR1 (Combined) GFTFSNFGMH SEQ ID NO: 18HC CDR2 (Combined) YISSGSSTIYYADTLKG SEQ ID NO: 19 HC CDR3 (Combined)RGEGAMDY SEQ ID NO: 25 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAYISSGSSTIYYADTLKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARRGEGAMDYWGQGTTVTVSS SEQ ID NO: 33 DNA VH CAGGTGCAGCTGGTGGAATCTGGTGGCGGAGTTGTGCAGCCTGGCAGATCCCTGAGACTGTCTTGTGC CGCCTCTGGCTTCACCTTCTCCAACTTCGGCATGCACTGGGTCCGACAGGCCCCTGGAAAAGGATTGG AGTGGGTCGCCTACATCTCCTCCGGCTCCTCCACCATCTACTACGCTGACACCCTGAAGGGCAGATTC ACCATCAGCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACAC CGCCGTGTACTACTGTGCTAGAAGAGGCGAGGGCGCCATGGATTATTGGGGCCAGGGAACCACAGTGA CCGTGTCTAGC Antibody B-H.1D LC-1SEQ ID NO: 20 LC CDR1 (Combined) RASSSVNYIY SEQ ID NO: 21LC CDR2 (Combined)) YTSNLAP SEQ ID NO: 22 LC CDR3 (Combined) QQFTSSPFTSEQ ID NO: 26 VL DNQLTQSPSFLSASVGDRVTITCRASSSVNYIYWYQQKPGKAPKLLIYYTSNLAPGVPSRFSGSGSGN EYTLTISSLQPEDFATYYCQQFTSSPFTFGQGTKLEIK SEQ ID NO: 34 DNA VL GATAACCAGCTGACCCAGTCTCCTAGCTTCCTGTCTGCCTCTGTGGGCGACAGAGTGACAATTACCTG CCGGGCCTCCTCCTCCGTGAACTACATCTACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGC TGATCTACTACACCTCCAATCTGGCCCCTGGCGTGCCCTCTAGATTTTCCGGATCTGGCTCCGGCAAC GAGTATACCCTGACAATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGTTCAC CTCCTCTCCATTCACCTTTGGCCAGGGCACCAAGCTGGAAATCAAA Antibody B-H.1E LC-2 SEQ ID NO: 20 LC CDR1 (Combined)RASSSVNYIY SEQ ID NO: 21 LC CDR2 (Combined)) YTSNLAP SEQ ID NO: 22LC CDR3 (Combined) QQFTSSPFT SEQ ID NO: 27 VLDNQLTQSPSSLSASVGDRVTITCRASSSVNYIYW YQQKPGKAPKLLIYYTSNLAPGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCQQFTSSPFTFGQGTK LEIK SEQ ID NO: 35 DNA VLATAACCAGCTGACCCAGTCTCCTTCCAGCCTGTC TGCTTCTGTGGGCGACAGAGTGACAATTACCTGCCGGGCCTCCTCCTCCGTGAACTACATCTACTGGT ATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACTACACCTCCAATCTGGCCCCTGGCGTG CCCTCTAGATTTTCCGGATCTGGCTCCGGCAACGACTATACCCTGACAATCTCCAGCCTGCAGCCTGA GGACTTCGCCACCTACTACTGCCAGCAGTTCACCTCCTCTCCATTCACCTTTGGCCAGGGCACCAAGC TGGAAATCAAA Antibody B-H.1F LC-3SEQ ID NO: 20 LC CDR1 (Combined) RASSSVNYIY SEQ ID NO: 21LC CDR2 (Combined)) YTSNLAP SEQ ID NO: 22 LC CDR3 (Combined) QQFTSSPFTSEQ ID NO: 28 VL ENVLTQSPATLSVSPGERATLSCRASSSVNYIYWYQQKPGQAPRLLIYYTSNLAPGIPARFSGSGSGN EYTLTISSLQSEDFAVYYCQQFTSSPFTFGQGTKLEIK SEQ ID NO: 36 DNA VL GAGAATGTGCTGACCCAGTCTCCTGCCACACTGTCTGTTAGCCCTGGCGAGAGAGCTACCCTGAGCTG CAGAGCCTCTTCCTCCGTGAACTACATCTACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTAGACTGC TGATCTACTACACCTCCAATCTGGCCCCTGGCATCCCTGCCAGATTTTCCGGATCTGGCTCCGGCAAC GAGTATACCCTGACCATCTCCAGCCTGCAGTCCGAGGACTTTGCTGTGTACTATTGCCAGCAGTTCAC AAGCAGCCCTTTCACCTTTGGCCAGGGCACCAAGCTGGAAATCAAA Antibody B-H.1G LC-4 SEQ ID NO: 20 LC CDR1 (Combined)RASSSVNYIY SEQ ID NO: 21 LC CDR2 (Combined)) YTSNLAP SEQ ID NO: 22LC CDR3 (Combined) QQFTSSPFT SEQ ID NO: 29 VLQNVLTQPPSASGTPGQRVTISCRASSSVNYIYWY QQLPGTAPKLLIYYTSNLAPGVPDRFSGSGSGNSYSLAISGLRSEDEADYYCQQFTSSPFTFGTGTKV TVL SEQ ID NO: 37 DNA VLCAGAATGTGCTGACCCAACCTCCTTCCGCCTCTG GCACACCTGGACAGAGAGTGACAATCTCCTGCCGGGCCTCCTCCTCCGTGAACTACATCTACTGGTAT CAGCAGCTGCCCGGCACCGCTCCTAAACTGCTGATCTACTACACCTCCAATCTGGCCCCTGGCGTGCC CGATAGATTTTCCGGATCTGGCTCCGGCAACTCCTACAGCCTGGCTATCTCTGGCCTGAGATCTGAGG ACGAGGCCGACTACTACTGCCAGCAGTTCACCTCCTCTCCATTCACCTTTGGCACCGGCACCAAAGTG ACAGTTCTT Antibody B-H.1H LC-5SEQ ID NO: 20 LC CDR1 (Combined) RASSSVNYIY SEQ ID NO: 21LC CDR2 (Combined)) YTSNLAP SEQ ID NO: 22 LC CDR3 (Combined) QQFTSSPFTSEQ ID NO: 30 VL SNELTQPPSVSVSPGQTARITCRASSSVNYIYWYQQKSGQAPVLVIYYTSNLAPGIPERFSGSGSGNM YTLTISGAQVEDEADYYCQQFTSSPFTFGTGTKVTVL SEQ ID NO: 38 DNA VL TCTAATGAGCTGACCCAGCCTCCTTCCGTGTCCGTGTCTCCTGGACAGACCGCCAGAATTACCTGCCG GGCCTCCTCCTCCGTGAACTACATCTACTGGTATCAGCAGAAGTCCGGCCAGGCTCCTGTGCTCGTGA TCTACTACACCTCCAATCTGGCCCCTGGCATCCCTGAGAGATTCTCCGGATCTGGCTCCGGCAACATG TACACCCTGACCATCTCTGGCGCCCAGGTGGAAGATGAGGCCGACTACTACTGCCAGCAGTTCACCTC CTCTCCATTCACCTTTGGCACCGGCACCAAAGTGACAGTTCTT Antibody B-H.1 SEQ ID NO: Chain1: Fc onlyMETDTLLLWVLLLWVPGSTGDKTHTCPPCPAPEL 3280LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO: Chain2: humanizedMETDTLLLWVLLLWVPGSTGEVQLVESGGGLVQP 3281 B-H scFvGGSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWV SYISSGSSTIYYADTLKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGEGAMDYWGQGTTVTVS SGGGGSGGGGSGGGGSGGGGSDNQLTQSPSFLSASVGDRVTITCRASSSVNYIYWYQQKPGKAPKLLI YYTSNLAPGVPSRFSGSGSGNEYTLTISSLQPEDFATYYCQQFTSSPFTFGQGTKLEIKGGGGSDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTK NQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKGGGGSGGGGSGLNDIFEAQKIEWHE

TABLE 3 Constant region amino acid sequences of human IgGheavy chains and human kappa light chain Human kappa LCRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ constant regionSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS  SEQ ID NO: 39PVTKSFNRGEC  IgG4 (S228P) HCASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS mutant constantGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK  region (EURVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV  Numbering)DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD  SEQ ID NO: 40WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG IgG1 wild type HCASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS SEQ ID NO: 41GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG1 (N297A) HCASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS mutant constantGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK region (EURVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC Numbering)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL SEQ ID NO: 42HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgM constant HCGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNS delta CDCDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGN (P311A, P313S)KEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQI SEQ ID NO: 73QVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKS TGKPTLYNVSLVMSDTAGTCYIgGA1 HC ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGV SEQ ID NO: 74TARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQD VTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKP THVNVSVVMAEVDGTCY IgGA2HC ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNV SEQ ID NO: 75TARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNSSQDVTVPCRVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTYAVTSILRVAAEDWKKGETFSCMVGHEALPLAFTQKTIDRMAGKPTHINVSVVMAEAD GTCY Human Ig_J HCMKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSS chainEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDP  SEQ ID NO: 76TEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGET  KMVETALTPDACYPD

Anti-TCRβ V5 Antibodies

Accordingly, in one aspect, the disclosure provides an anti-TCRβVantibody molecule that binds to human TCRβ V5. In some embodiments, theTCRβ V5 subfamily comprises TCRβ V5-5*01, TCRβ V5-6*01, TCRβ V5-4*01,TCRβ V5-8*01, TCRβ V5-1*01, or a variant thereof.

TABLE 10 Amino acid sequences for anti TCRβ V5 antibodiesMurine antibody C SEQ ID NO: 232 VHDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPRT FGGGTKVEIKSEQ ID NO: 233 VL QVQLKESGPGLVAPSQSLSITCTVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARDRVTATLYAMDYWGQGTSVTVSS Humanized antibody C (C-H antibody)Variable light chain (VL) SEQ ID NO: 3000 VL C-H.1DIQMTQSPSFLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSSLHSGVPSRFSGSGSGTEYTLTISSLQPED FATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3001 VL C-H.2 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3002 VL C-H.3 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVVKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPED VATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3003 VL C-H.4 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGQAVKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPED VATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3004 VL C-H.5 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSSLHSGVPSRFSGSGSGTDYTFTISSLQPED IATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3005 VL C-H.6 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKTVKLLIYYTSSLHSGIPSRFSGSGSGTDYTLTIRSLQPED FATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3006 VL C-H.7 AIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3007 VL C-H.8 DIQMTQSPSSVSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3008 VL C-H.9 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKRLIYYTSSLHSGVPSRFSGSGSGTEYTLTISNLQPE DFATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3009 VL C-H.10 AIRMTQSPFSLSASVGDRVTITCSASQGISNYLNWYQQKPAKAVKLFIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3010 VL C-H.11 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKRLIYYTSSLHSGVPSRFSGSGSGTEYTLTISSLQPED FATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3011 VL C-H.12 DIQMTQSPSTLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSSLHSGVPSRFSGSGSGTEYTLTISSLQPDD FATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3012 VL C-H.13 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKSLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3013 VL C-H.14 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKSLIYYTSSLHSGVPSKFSGSGSGTDYTLTISSLQPED FATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3014 VL C-H.15 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPEKAVKSLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDF ATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3015 VL C-H.16 DIQMTQSPSAMSASVGDRVTITCSASQGISNYLNWYQQKPGKVVKRLIYYTSSLHSGVPSRFSGSGSGTEYTLTISSLQPED FATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3016 VL C-H.17 DIVMTQSPDSLAVSLGERATINCSASQGISNYLNWYQQKPGQPVKLLIYYTSSLHSGVPDRFSGSGSGTDYTLTISSLQAE DVAVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3017 VL C-H.18 EIVMTQSPGTLSLSPGERATLSCSASQGISNYLNWYQQKPGQAVKLLIYYTSSLHSGIPDRFSGSGSGTDYTLTISRLEPED FAVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3018 VL C-H.19 EIVMTQSPPTLSLSPGERVTLSCSASQGISNYLNWYQQKPGQAVKLLIYYTSSLHSGIPARFSGSGSGTDYTLTISSLQPEDF AVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3019 VL C-H.20 EIVMTQSPPTLSLSPGERVTLSCSASQGISNYLNWYQQKPGQAVKLLIYYTSSLHSSIPARFSGSGSGTDYTLTISSLQPEDF AVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3020 VL C-H.21 EIVMTQSPATLSLSPGERATLSCSASQGISNYLNWYQQKPGQAVKLLIYYTSSLHSGIPARFSGSGSGTDYTLTISSLEPED FAVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3021 VL C-H.22 EIVMTQSPATLSLSPGERATLSCSASQGISNYLNWYQQKPGQAVKLLIYYTSSLHSGIPARFSGSGSGTDYTLTISRLEPED FAVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3022 VL C-H.23 EIVMTQSPATLSLSPGERATLSCSASQGISNYLNWYQQKPGQAVKLLIYYTSSLHSGIPDRFSGSGSGTDYTLTISRLEPED FAVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3023 VL C-H.24 EIVMTQSPATLSLSPGERATLSCSASQGISNYLNWYQQKPGLAVKLLIYYTSSLHSGIPDRFSGSGSGTDYTLTISRLEPED FAVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3024 VL C-H.25 DIQMIQSPSFLSASVGDRVSIICSASQGISNYLNWYLQKPGKSVKLFIYYTSSLHSGVSSRFSGRGSGTDYTLTIISLKPEDF AAYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3025 VL C-H.26 EIVMTQSPATLSLSPGERATLSCSASQGISNYLNWYQQKPGQAVKLLIYYTSSLHSGIPARFSGSGSGTDYTLTISSLQPED FAVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3026 VL C-H.27 EIVMTQSPATLSLSPGERATLSCSASQGISNYLNWYQQKPGQAVKLLIYYTSSLHSGIPARFSGSGPGTDYTLTISSLEPED FAVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3027 VL C-H.28 DIVMTQTPLSLSVTPGQPASISCSASQGISNYLNWYLQKPGQSVKLLIYYTSSLHSGVPDRFSGSGSGTDYTLKISRVEAED VGVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3028 VL C-H.29 DIVMTQTPLSLSVTPGQPASISCSASQGISNYLNWYLQKPGQPVKLLIYYTSSLHSGVPDRFSGSGSGTDYTLKISRVEAED VGVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3029 VL C-H.30 DIVMTQSPAFLSVTPGEKVTITCSASQGISNYLNWYQQKPDQAVKLLIYYTSSLHSGVPSRFSGSGSGTDYTFTISSLEAED AATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3030 VL C-H.31 DIVMTQSPLSLPVTPGEPASISCSASQGISNYLNWYLQKPGQSVKLLIYYTSSLHSGVPDRFSGSGSGTDYTLKISRVEAED VGVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3031 VL C-H.32 DIVMTQTPLSLPVTPGEPASISCSASQGISNYLNWYLQKPGQSVKLLIYYTSSLHSGVPDRFSGSGSGTDYTLKISRVEAED VGVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3032 VL C-H.33 EIVMTQSPATLSVSPGERATLSCSASQGISNYLNWYQQKPGQAVKLLIYYTSSLHSGIPARFSGSGSGTEYTLTISILQSEDF AVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3033 VL C-H.34 EIVMTQSPATLSVSPGERATLSCSASQGISNYLNWYQQKPGQAVKLLIYYTSSLHSGIPARFSGSGSGTEYTLTISSLQSED FAVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3034 VL C-H.35 DIVMTQSPLSLPVTLGQPASISCSASQGISNYLNWYQQRPGQSVKRLIYYTSSLHSGVPDRFSGSGSGTDYTLKISRVEAED VGVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3035 VL C-H.36 EITMTQSPAFMSATPGDKVNISCSASQGISNYLNWYQQKPGEAVKFIIYYTSSLHSGIPPRFSGSGYGTDYTLTINNIESEDA AYYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3036 VL C-H.37 DIVMTQTPLSSPVTLGQPASISCSASQGISNYLNWYQQRPGQPVKLLIYYTSSLHSGVPDRFSGSGAGTDYTLKISRVEAED VGVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3037 VL C-H.38 EIVMTQSPDFQSVTPKEKVTITCSASQGISNYLNWYQQKPDQSVKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTINSLEAE DAATYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3038 VL C-H.39 EIVMTQTPLSLSITPGEQASISCSASQGISNYLNWYLQKARPVVKLLIYYTSSLHSGVPDRFSGSGSGTDYTLKISRVEAEDF GVYYCQQYSKLPRTFGGGTKVEIKSEQ ID NO: 3039 VL C-H.40 EIVMTQTPLSLSITPGEQASMSCSASQGISNYLNWYLQKARPVVKLLIYYTSSLHSGVPDRFSGSGSGTDYTLKISRVEAED FGVYYCQQYSKLPRTFGGGTKVEIKVariable HEAVY chain (VH) SEQ ID NO: 3040 VH C-H.1QVTLKESGPVLVKPTETLTLTCTVSGFSLTAYGVNWVRQPPGKALEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVVLTMTNMDPVDTATYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3041 VH C-H.2QVTLKESGPALVKPTETLTLTCTVSGFSLTAYGVNWVRQPPGKALEWLGMIWGDGNTDYNSALKSRLIISKDNSKSQVVLTMTNMDPVDTATYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3042 VH C-H.3QVTLKESGPALVKPTQTLTLTCTVSGFSLTAYGVNWVRQPPGKALEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVVLTMTNMDPVDTATYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3043 VH C-H.4QVQLQESGPGLVKPSGTLSLTCAVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3044 VH C-H.5QVTLKESGPTLVKPTQTLTLTCTVSGFSLTAYGVNWVRQPPGKALEWLGMIWGDGNTDYNSALKSRLTITKDNSKSQVVLTMTNMDPVDTATYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3045 VH C-H.6QVTLKESGPALVKPTQTLTLTCTVSGFSLTAYGVNWVRQPPGKALEWLGMIWGDGNTDYNSALKSRLTITKDNSKSQVVLTMTNMDPVDTATYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3046 VH C-H.7QVQLQESGPGLVKPSQTLSLTCTVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLVT VSS SEQ ID NO: 3047 VH C-H.8QVQLQESGPGLVKPSETLSLTCTVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLVT VSS SEQ ID NO: 3048 VH C-H.9QVQLQESGPGLVKPSQTLSLTCAVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3049 VH C-H.10QVQLQESGPGLVKPSDTLSLTCTVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLVT VSS SEQ ID NO: 3050 VH C-H.11QVQLQESGPGLVKPSQTLSLTCTVSGFSLTAYGVNWVRQHPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3051 VH C-H.12QVQLQESGPGLVKPSQTLSLTCTVSGFSLTAYGVNWVRQPAGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLVT VSS SEQ ID NO: 3052 VH C-H.13QVQLQESGPGLVKPSQTLSLTCAVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAVDTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3053 VH C-H.14QVQLQESGPGLVKPSETLSLTCTVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSHVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLVT VSS SEQ ID NO: 3054 VH C-H.15QVQLQESGPGLVKPSETLSLTCAVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLVT VSS SEQ ID NO: 3055 VH C-H.16QVQLQESGPGLVKPSQTLSLTCAVYGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3056 VH C-H.17RVQLQESGPGLVKPSETLSLTCTVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVPLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLVT VSS SEQ ID NO: 3057 VH C-H.18QVQLQESGPGLVKPSQTLSLTCTVSGFSLTAYGVNWVRQHPGKGLEWLGMIWGDGNTDYNSALKSLLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3058 VH C-H.19QVQLQESGPGLVKPSDTLSLTCAVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTALDTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3059 VH C-H.20QVQLQESGPGLVKPSDTLSLTCAVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAVDTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3060 VH C-H.21QVQLQESGSGLVKPSQTLSLTCAVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3061 VH C-H.22EVQLVESGGGLVQPGRSLRLSCTVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSIVYLQMNSLKTEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3062 VH C-H.23EVQLVESGGGLVQPGPSLRLSCTVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSIVYLQMNSLKTEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3063 VH C-H.24QVQLQESGSGLVKPSQTLSLTCAVSGFSLTAYGVNWVRQSPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3064 VH C-H.25QVQLQESGPGLVKPSETLSLTCTVSGFSLTAYGVNWVRQPAGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLVT VSS SEQ ID NO: 3065 VH C-H.26EVQLVESGGGLVKPGRSLRLSCTVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSIVYLQMNSLKTEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3066 VH C-H.27QVQLQESGPGLVKPSETLSLTCAVYGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVYLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3067 VH C-H.28QVQLQESGPGLVKPSDTLSLTCAVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAVDTGVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3068 VH C-H.29EVQLVESGGGLVQPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSSVYLQMNSLKTEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3069 VH C-H.30EVQLVESGGGLVKPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSTVYLQMNSLKTEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3070 VH C-H.31QVQLQQSGPGLVKPSQTLSLTCAVSGFSLTAYGVNWVRQSPSRGLEWLGMIWGDGNTDYNSALKSRLTINKDNSKSQVSLQLNSVTPEDTAVYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3071 VH C-H.32QVQLVESGGGLVQPGGSLRLSCSVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSTVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3072 VH C-H.33QVQLQQWGAGLLKPSETLSLTCAVYGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3073 VH C-H.34QVQLVESGGGVVQPGRSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSTSTVFLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3074 VH C-H.35EVQLVESGGGLVQPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSTVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3075 VH C-H.36EVQLVESGGGLVQPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNAKSSVYLQMNSLRDEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3076 VH C-H.37EVQLLESGGGLVQPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSTVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3077 VH C-H.38QVQLVESGGGLVKPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNAKSSVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3078 VH C-H.39EVQLVESGGGLVQPGGSLKLSCAVSGFSLTAYGVNWVRQASGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSTVYLQMNSLKTEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3079 VH C-H.40QVQLLESGGGLVKPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNAKSSVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3080 VH C-H.41QVQLVESGGGVVQPGRSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSTVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3081 VH C-H.42QVQLVESGGGVVQPGRSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSRVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3082 VH C-H.43QVQLVESGGGVVQPGRSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLAISKDNSKSTVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3083 VH C-H.44QVQLVESGGGVVQPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSTVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3084 VH C-H.45EVQLVESGGGLVQPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNAKSTVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3085 VH C-H.46EVQLVESGGGLVQPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNAKSSVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3086 VH C-H.47EVQLVESGGVVVQPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSSVYLQMNSLRTEDTALYYCARDRVTATLYAMDYWGQGTLV TVSS SEQ ID NO: 3087 VH C-H.48EVQLVESGGGLVQPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKHNSKSTVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3088 VH C-H.49EVQLVESGGGLVKPGGSLRLSCAVSGFSLTAYGVNWVRQAPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNAKSSVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTL VTVSS SEQ ID NO: 3089 VH C-H.50EVQLVESGGGLIQPGGSLRLSCAVSGFSLTAYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLTISKDNSKSTVYLQMNSLRAEDTAVYYCARDRVTATLYAMDYWGQGTLV TVSS

Antibody E comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 3284 and a light chain comprising the amino acid sequence ofSEQ ID NO: 3285.

TABLE 11 Amino acid sequences for anti TCRβ V5 antibodiesMurine antibody E SEQ ID NO: 3091 VHQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMHLSSLTSVDSAVYFCARRGTGGWYFDVWGAGTTVTVSS SEQ ID NO: 3284 HeavyMETDTLLLWVLLLWVPGSTGQVQLQQSGPELVKPGASVKISCK chainASGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMHLSSLTSVDSAVYFCARRGTGGWYFDVWGAGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 3092 VLDIVLTQSPASLAVSLGQRATISCRASESVDSSGNSFMHWYQQKPGQPPQLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATF YCQQSFDDPFTFGSGTKLEIKSEQ ID NO: 3285 Light METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCR chainASESVDSSGNSFMHWYQQKPGQPPQLLIYRASNLESGIPARFSGSGSRTDFTLTTNPVEADDVATFYCQQSFDDPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATH KTSTSPIVKSFNRNECHumanized antibody E (E-H antibody) Variable light chain (VL)SEQ ID NO: 3093 VL E-H.1 DIVLTQSPDSLAVSLGERATINCRASESVDSSGNSFMHWYQQKPGQPPQLLIYRASNLESGVPDRFSGSGSRTDFTLTISS LQAEDVAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3094 VL E-H.2 EIVLTQSPATLSLSPGERATLSCRASESVDSSGNSFMHWYQQKPGQAPQLLIYRASNLESGIPARFSGSGSRTDFTLTISS LEPEDFAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3095 VL E-H.3 EIVLTQSPATLSLSPGERATLSCRASESVDSSGNSFMHWYQQKPGQAPQLLIYRASNLESGIPARFSGSGSRTDFTLTISR LEPEDFAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3096 VL E-H.4 EIVLTQSPATLSLSPGERATLSCRASESVDSSGNSFMHWYQQKPGQAPQLLIYRASNLESGIPARFSGSGSRTDFTLTISS LQPEDFAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3097 VL E-H.5 DIQLTQSPSSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGQAPQLLIYRASNLESGVPSRFSGSGSRTDFTLTISS LQPEDVATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3098 VL E-H.6 EIVLTQSPATLSLSPGERATLSCRASESVDSSGNSFMHWYQQKPGQAPQLLIYRASNLESGIPARFSGSGPRTDFTLTISS LEPEDFAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3099 VL E-H.7 EIVLTQSPATLSLSPGERATLSCRASESVDSSGNSFMHWYQQKPGQAPQLLIYRASNLESGIPDRFSGSGSRTDFTLTISR LEPEDFAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3100 VL E-H.8 DIQLTQSPSSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKVPQLLIYRASNLESGVPSRFSGSGSRTDFTLTISS LQPEDVATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3101 VL E-H.9 DIQLTQSPSSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKTPQLLIYRASNLESGIPSRFSGSGSRTDFTLTIRSL QPEDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3102 VL E-H.10 EIVLTQSPGTLSLSPGERATLSCRASESVDSSGNSFMHWYQQKPGQAPQLLIYRASNLESGIPDRFSGSGSRTDFTLTISR LEPEDFAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3103 VL E-H.11 EIVLTQSPATLSLSPGERATLSCRASESVDSSGNSFMHWYQQKPGLAPQLLIYRASNLESGIPDRFSGSGSRTDFTLTISR LEPEDFAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3104 VL E-H.12 DIQLTQSPSSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKAPQLLIYRASNLESGVPSRFSGSGSRTDFTLTISS LQPEDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3105 VL E-H.13 DIQLTQSPSSVSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKAPQLLIYRASNLESGVPSRFSGSGSRTDFTLTISS LQPEDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3106 VL E-H.14 AIQLTQSPSSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKAPQLLIYRASNLESGVPSRFSGSGSRTDFTLTISS LQPEDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3107 VL E-H.15 DIQLTQSPSFLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKAPQLLIYRASNLESGVPSRFSGSGSRTEFTLTISS LQPEDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3108 VL E-H.16 DIQLTQSPSSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKAPQLLIYRASNLESGVPSRFSGSGSRTDFTFTISS LQPEDIATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3109 VL E-H.17 EIVLTQSPATLSVSPGERATLSCRASESVDSSGNSFMHWYQQKPGQAPQLLIYRASNLESGIPARFSGSGSRTEFTLTISIL QSEDFAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3110 VL E-H.18 EIVLTQSPATLSVSPGERATLSCRASESVDSSGNSFMHWYQQKPGQAPQLLIYRASNLESGIPARFSGSGSRTEFTLTISSL QSEDFAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3111 VL E-H.19 AIRLTQSPFSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPAKAPQLFIYRASNLESGVPSRFSGSGSRTDFTLTISS LQPEDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3112 VL E-H.20 DIQLTQSPSSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKAPQSLIYRASNLESGVPSRFSGSGSRTDFTLTISS LQPEDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3113 VL E-H.21 DIQLTQSPSSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKAPQRLIYRASNLESGVPSRFSGSGSRTEFTLTISN LQPEDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3114 VL E-H.22 DIQLTQSPSTLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKAPQLLIYRASNLESGVPSRFSGSGSRTEFTLTISS LQPDDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3115 VL E-H.23 EIVLTQSPDFQSVTPKEKVTITCRASESVDSSGNSFMHWYQQKPDQSPQLLIYRASNLESGVPSRFSGSGSRTDFTLTINS LEAEDAATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3116 VL E-H.24 DIQLTQSPSSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKAPQSLIYRASNLESGVPSKFSGSGSRTDFTLTISS LQPEDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3117 VL E-H.25 DIQLTQSPSSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKAPQRLIYRASNLESGVPSRFSGSGSRTEFTLTISS LQPEDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3118 VL E-H.26 DIVLTQTPLSLSVTPGQPASISCRASESVDSSGNSFMHWYLQKPGQPPQLLIYRASNLESGVPDRFSGSGSRTDFTLKISR VEAEDVGVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3119 VL E-H.27 DIQLTQSPSSLSASVGDRVTITCRASESVDSSGNSFMHWYQQKPEKAPQSLIYRASNLESGVPSRFSGSGSRTDFTLTISS LQPEDFATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3120 VL E-H.28 EIVLTQSPPTLSLSPGERVTLSCRASESVDSSGNSFMHWYQQKPGQAPQLLIYRASNLESGIPARFSGSGSRTDFTLTISS LQPEDFAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3121 VL E-H.29 DIQLTQSPSAMSASVGDRVTITCRASESVDSSGNSFMHWYQQKPGKVPQRLIYRASNLESGVPSRFSGSGSRTEFTLTISSLQPEDFATYYCQQSFDDPFTFGQGTKLEIK SEQ ID NO: 3122 VL E-H.30DIVLTQSPLSLPVTPGEPASISCRASESVDSSGNSFMHWYLQKPGQSPQLLIYRASNLESGVPDRFSGSGSRTDFTLKISRV EAEDVGVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3123 VL E-H.31 DIVLTQTPLSLPVTPGEPASISCRASESVDSSGNSFMHWYLQKPGQSPQLLIYRASNLESGVPDRFSGSGSRTDFTLKISRV EAEDVGVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3124 VL E-H.32 DIVLTQTPLSLSVTPGQPASISCRASESVDSSGNSFMHWYLQKPGQSPQLLIYRASNLESGVPDRFSGSGSRTDFTLKISR VEAEDVGVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3125 VL E-H.33 EIVLTQSPPTLSLSPGERVTLSCRASESVDSSGNSFMHWYQQKPGQAPQLLIYRASNLESSIPARFSGSGSRTDFTLTISSL QPEDFAVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3126 VL E-H.34 DIVLTQSPLSLPVTLGQPASISCRASESVDSSGNSFMHWYQQRPGQSPQRLIYRASNLESGVPDRFSGSGSRTDFTLKISR VEAEDVGVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3127 VL E-H.35 DIVLTQTPLSSPVTLGQPASISCRASESVDSSGNSFMHWYQQRPGQPPQLLIYRASNLESGVPDRFSGSGARTDFTLKISR VEAEDVGVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3128 VL E-H.36 DIVLTQSPAFLSVTPGEKVTITCRASESVDSSGNSFMHWYQQKPDQAPQLLIYRASNLESGVPSRFSGSGSRTDFTFTISS LEAEDAATYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3129 VL E-H.37 DIQLIQSPSFLSASVGDRVSIICRASESVDSSGNSFMHWYLQKPGKSPQLFIYRASNLESGVSSRFSGRGSRTDFTLTIISLK PEDFAAYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3130 VL E-H.38 EIVLTQTPLSLSITPGEQASISCRASESVDSSGNSFMHWYLQKARPVPQLLIYRASNLESGVPDRFSGSGSRTDFTLKISRV EAEDFGVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3131 VL E-H.39 EIVLTQTPLSLSITPGEQASMSCRASESVDSSGNSFMHWYLQKARPVPQLLIYRASNLESGVPDRFSGSGSRTDFTLKISR VEAEDFGVYYCQQSFDDPFTFGQGTKLEIKSEQ ID NO: 3132 VL E-H.40 EITLTQSPAFMSATPGDKVNISCRASESVDSSGNSFMHWYQQKPGEAPQFIIYRASNLESGIPPRFSGSGYRTDFTLTINNI ESEDAAYYYCQQSFDDPFTFGQGTKLEIKVariable HEAVY chain (VH) SEQ ID NO: 3133 VH E-H.1QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWVRQAPGQGLEWIGRIYPGDGDTKYNGKFKGRATLTADKSTSTAYMELSSLRSEDTAVYYCARRGTGGWYFDVWGQG TTVTVSS SEQ ID NO: 3134 VH E-H.2QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSSWMNWVRQAPGQGLEWIGRIYPGDGDTKYNGKFKGRATLTADKSTSTAYMELSSLRSEDTAVYYCARRGTGGWYFDVWGQG TTVTVSS SEQ ID NO: 3135 VH E-H.3QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLTADKSTSTAYMELSSLRSEDTAVYYCARRGTGGWYFDVWGQG TTVTVSS SEQ ID NO: 3136 VH E-H.4QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWVRQAPGQELEWIGRIYPGDGDTKYNGKFKGRATLTADKSISTAYMELSSLRSEDTATYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3137 VH E-H.5EVQLVQSGAEVKKPGATVKISCKASGYAFSSSWMNWVQQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLTADKSTSTAYMELSSLRSEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3138 VH E-H.6QVQLVQSGAEVKKTGSSVKVSCKASGYAFSSSWMNWVRQAPGQALEWIGRIYPGDGDTKYNGKFKGRATLTADKSMSTAYMELSSLRSEDTAMYYCARRGTGGWYFDVWGQG TTVTVSS SEQ ID NO: 3139 VH E-H.7QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWVRQAPGQRLEWIGRIYPGDGDTKYNGKFKGRATLTADKSASTAYMELSSLRSEDMAVYYCARRGTGGWYFDVWGQG TTVTVSS SEQ ID NO: 3140 VH E-H.8QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWVRQAPGQGLEWIGRIYPGDGDTKYNGKFKGRATLTADKSTSTAYMELRSLRSDDMAVYYCARRGTGGWYFDVWGQG TTVTVSS SEQ ID NO: 3141 VH E-H.9QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWVRQAPGQRLEWIGRIYPGDGDTKYNGKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARRGTGGWYFDVWGQG TTVTVSS SEQ ID NO: 3142 VH E-H.10QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWVRQAPGQGLEWIGRIYPGDGDTKYNGKFKGRATLTADKSTSTAYMELRSLRSDDTAVYYCARRGTGGWYFDVWGQG TTVTVSS SEQ ID NO: 3143 VH E-H.11QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWVRQAPGQGLEWIGRIYPGDGDTKYNGKFKGRATLTADKSISTAYMELSRLRSDDTAVYYCARRGTGGWYFDVWGQGT TVTVSS SEQ ID NO: 3144 VH E-H.12QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWVRQAPGQGLEWIGRIYPGDGDTKYNGKFKGRATLTADKSISTAYMELSRLRSDDTVVYYCARRGTGGWYFDVWGQGT TVTVSS SEQ ID NO: 3145 VH E-H.13QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWVRQAPGQGLEWIGRIYPGDGDTKYNGKFKGWATLTADKSISTAYMELSRLRSDDTAVYYCARRGTGGWYFDVWGQGT TVTVSS SEQ ID NO: 3146 VH E-H.14QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWVRQATGQGLEWIGRIYPGDGDTKYNGKFKGRATLTANKSISTAYMELSSLRSEDTAVYYCARRGTGGWYFDVWGQGT TVTVSS SEQ ID NO: 3147 VH E-H.15QVQLVQSGSELKKPGASVKVSCKASGYAFSSSWMNWVRQAPGQGLEWIGRIYPGDGDTKYNGKFKGRAVLSADKSVSTAYLQISSLKAEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3148 VH E-H.16QVQLVQSGPEVKKPGTSVKVSCKASGYAFSSSWMNWVRQARGQRLEWIGRIYPGDGDTKYNGKFKGRATLTADKSTSTAYMELSSLRSEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3149 VH E-H.17EVQLVQSGAEVKKPGESLKISCKASGYAFSSSWMNWVRQMPGKGLEWIGRIYPGDGDTKYNGKFKGQATLSADKSISTAYLQWSSLKASDTAMYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3150 VH E-H.18QVQLVQSGSELKKPGASVKVSCKASGYAFSSSWMNWVRQAPGQGLEWIGRIYPGDGDTKYNGKFKGRAVLSADKSVSMAYLQISSLKAEDTAVYYCARRGTGGWYFDVWGQGT TVTVSS SEQ ID NO: 3151 VH E-H.19QVQLVQSGHEVKQPGASVKVSCKASGYAFSSSWMNWVPQAPGQGLEWIGRIYPGDGDTKYNGKFKGRAVLSADKSASTAYLQISSLKAEDMAMYYCARRGTGGWYFDVWGQG TTVTVSS SEQ ID NO: 3152 VH E-H.20EVQLVQSGAEVKKPGESLKISCKASGYAFSSSWMNWVRQMPGKGLEWIGRIYPGDGDTKYNGKFKGQATLSADKPISTAYLQWSSLKASDTAMYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3153 VH E-H.21EVQLVQSGAEVKKPGESLRISCKASGYAFSSSWMNWVRQMPGKGLEWIGRIYPGDGDTKYNGKFKGQATLSADKSISTAYLQWSSLKASDTAMYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3154 VH E-H.22EVQLVQSGAEVKKPGESLRISCKASGYAFSSSWMNWVRQMPGKGLEWIGRIYPGDGDTKYNGKFKGHATLSADKSISTAYLQWSSLKASDTAMYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3155 VH E-H.23QVQLVQSGAEVKKTGSSVKVSCKASGYAFSSSWMNWVRQAPRQALEWIGRIYPGDGDTKYNGKFKGRATLTADKSMSTAYMELSSLRSEDTAMYYCARRGTGGWYFDVWGQG TTVTVSS SEQ ID NO: 3156 VH E-H.24EVQLVESGGGLVQPGRSLRLSCTASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSIAYLQMNSLKTEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3157 VH E-H.25EVQLVESGGGLVQPGPSLRLSCTASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSIAYLQMNSLKTEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3158 VH E-H.26QVQLQESGPGLVKPSQTLSLTCTASGYAFSSSWMNWVRQPPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSQASLKLSSVTAADTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3159 VH E-H.27QVQLQESGPGLVKPSGTLSLTCAASGYAFSSSWMNWVRQPPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSQASLKLSSVTAADTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3160 VH E-H.28EVQLVESGGGLVKPGRSLRLSCTASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSIAYLQMNSLKTEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3161 VH E-H.29EVQLVESGGGLVQPGGSLKLSCAASGYAFSSSWMNWVRQASGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSTAYLQMNSLKTEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3162 VH E-H.30QVQLQESGPGLVKPSQTLSLTCAASGYAFSSSWMNWVRQPPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSQASLKLSSVTAADTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3163 VH E-H.31EVQLVESGGGLVKPGGSLRLSCAASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSTAYLQMNSLKTEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3164 VH E-H.32EVQLVESGGALVKPGGSLRLSCAASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSTAYLQMNSLKTEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3165 VH E-H.33QVQLQESGPGLVKPSQTLSLTCAAYGYAFSSSWMNWVRQPPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSQASLKLSSVTAADTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3166 VH E-H.34QVQLQESGSGLVKPSQTLSLTCAASGYAFSSSWMNWVRQPPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSQASLKLSSVTAADTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3167 VH E-H.35EVQLVESGGGLVQPGGSLRLSCAASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSSAYLQMNSLKTEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3168 VH E-H.36QVQLQESGPGLVKPSDTLSLTCTASGYAFSSSWMNWVRQPPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSQASLKLSSVTAADTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3169 VH E-H.37QVQLQESGPGLVKPSQTLSLTCTASGYAFSSSWMNWVRQHPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSQASLKLSSVTAADTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3170 VH E-H.38QVQLQESGPGLVKPSQTLSLTCTASGYAFSSSWMNWVRQHPGKGLEWIGRIYPGDGDTKYNGKFKGLATLSADKSKSQASLKLSSVTAADTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3171 VH E-H.39QVQLVESGGGVVQPGRSLRLSCAASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSTAYLQMSSLRAEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3172 VH E-H.40QVQLVESGGGLVKPGGSLRLSCAASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKAKSSAYLQMNSLRAEDTAVYYCARRGTGGWYFDVWGQGT TVTVSS SEQ ID NO: 3173 VH E-H.41QVQLVESGGGLVQPGGSLRLSCSASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSTAYLQMNSLRAEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3174 VH E-H.42QVQLLESGGGLVKPGGSLRLSCAASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKAKSSAYLQMNSLRAEDTAVYYCARRGTGGWYFDVWGQGT TVTVSS SEQ ID NO: 3175 VH E-H.43EVQLVESGGGLVQPGGSLRLSCSASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSTAYLQMSSLRAEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3176 VH E-H.44QVQLQESGPGLVKPSDTLSLTCAASGYAFSSSWMNWVRQPPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSQASLKLSSVTAVDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3177 VH E-H.45QVQLQESGPGLVKPSQTLSLTCAASGYAFSSSWMNWVRQPPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSQASLKLSSVTAVDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3178 VH E-H.46EVQLVESGGGLVQPGGSLRLSCSASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSTAYVQMSSLRAEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3179 VH E-H.47QVQLVDSGGGVVQPGRSLRLSCAASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSTAYLQMNSLRAEDTAVYYCARRGTGGWYFDVWGQG TTVTVSS SEQ ID NO: 3180 VH E-H.48QVQLVESGGGVVQPGRSLRLSCAASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSTAYLQMNSLRAEGTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3181 VH E-H.49QVQLVESGGGVVQPGRSLRLSCAASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSTAYLQMNSLRAEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS SEQ ID NO: 3182 VH E-H.50EVQLVESGGGLVQPGGSLRLSCAASGYAFSSSWMNWVRQAPGKGLEWIGRIYPGDGDTKYNGKFKGRATLSADKSKSTAYLQMNSLRAEDTAVYYCARRGTGGWYFDVWGQGTT VTVSS

In some embodiments, the anti-TCRβ V5 antibody molecule comprises a VHand/or a VL of an antibody described in Table 10, or a sequence with atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβ V5 antibody molecule comprises a VHand a VL of an antibody described in Table 10, or a sequence with atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβ V5 antibody molecule comprises a VHand/or a VL of an antibody described in Table 11, or a sequence with atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβ V5 antibody molecule comprises a VHand a VL of an antibody described in Table 11, or a sequence with atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

Anti-TCRβ V10 Antibodies

Accordingly, in one aspect, the disclosure provides an anti-TCRβVantibody molecule that binds to a human TCRβ V10 subfamily member. Insome embodiments, TCRβ V10 subfamily is also known as TCRβ V12. In someembodiments, the TCRβ V10 subfamily comprises: TCRβ V10-1*01, TCRβV10-1*02, TCRβ V10-3*01 or TCRβ V10-2*01, or a variant thereof.

TABLE 12 Amino acid sequences for anti TCRβ V10 antibodiesMurine antibody D SEQ ID NO: 3183 VHEVQLVESGGDLVKPGGSLKLSCAVSGFTFRSYGMSWVRQTPDKRLEWVALISSGGSYTYYTDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAIYYCSRHGGNFFDYWGQGTTLTVSS SEQ ID NO: 3184 VLQIVLTQSPSIMSASPGEKVTMTCSVSSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWS SNPQYTFGGGTKLEIKHumanized antibody D (D-H antibody) Variable light chain (VL)SEQ ID NO: 3185 VL D-H.1 DIVLTQSPAFLSVTPGEKVTITCSVSSSVSYMHWYQQKPDQAPKLLIYDTSKLASGVPSRFSGSGSGTDYTFTISSLEAED AATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3186 VL D-H.2 AIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3187 VL D-H.3 DIQLTQSPSFLSASVGDRVTITCSVSSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEYTLTISSLQPED FATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3188 VL D-H.4 DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3189 VL D-H.5 DIQLTQSPSSVSASVGDRVTITCSVSSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3190 VL D-H.6 DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQKPGKVPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPED VATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3191 VL D-H.7 DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQKPGQAPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPED VATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3192 VL D-H.8 EIVLTQSPDFQSVTPKEKVTITCSVSSSVSYMHWYQQKPDQSPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTINSLEAED AATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3193 VL D-H.9 AIRLTQSPFSLSASVGDRVTITCSVSSSVSYMHWYQQKPAKAPKLFIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3194 VL D-H.10 DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDI ATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3195 VL D-H.11 EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQKPGQAPKLLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDF AVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3196 VL D-H.12 DIQLTQSPSTLSASVGDRVTITCSVSSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEYTLTISSLQPDD FATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3197 VL D-H.13 DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQKPGKTPKLLIYDTSKLASGIPSRFSGSGSGTDYTLTIRSLQPEDF ATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3198 VL D-H.14 EIVLTQSPPTLSLSPGERVTLSCSVSSSVSYMHWYQQKPGQAPKLLIYDTSKLASGIPARFSGSGSGTDYTLTISSLQPED FAVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3199 VL D-H.15 DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISSLQPED FATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3200 VL D-H.16 EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQKPGQAPKLLIYDTSKLASGIPARFSGSGPGTDYTLTISSLEPEDF AVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3201 VL D-H.17 EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQKPGQAPKLLIYDTSKLASGIPARFSGSGSGTDYTLTISRLEPED FAVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3202 VL D-H.18 EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQKPGQAPKLLIYDTSKLASGIPARFSGSGSGTDYTLTISSLQPED FAVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3203 VL D-H.19 EIVLTQSPATLSVSPGERATLSCSVSSSVSYMHWYQQKPGQAPKLLIYDTSKLASGIPARFSGSGSGTEYTLTISSLQSEDF AVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3204 VL D-H.20 EIVLTQSPATLSVSPGERATLSCSVSSSVSYMHWYQQKPGQAPKLLIYDTSKLASGIPARFSGSGSGTEYTLTISILQSEDF AVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3205 VL D-H.21 EIVLTQSPPTLSLSPGERVTLSCSVSSSVSYMHWYQQKPGQAPKLLIYDTSKLASSIPARFSGSGSGTDYTLTISSLQPEDF AVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3206 VL D-H.22 DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQKPGKAPKSLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3207 VL D-H.23 DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISNLQPED FATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3208 VL D-H.24 DIQLTQSPSAMSASVGDRVTITCSVSSSVSYMHWYQQKPGKVPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISSLQPE DFATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3209 VL D-H.25 EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQKPGQAPKLLIYDTSKLASGIPDRFSGSGSGTDYTLTISRLEPED FAVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3210 VL D-H.26 EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQKPGLAPKLLIYDTSKLASGIPDRFSGSGSGTDYTLTISRLEPEDF AVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3211 VL D-H.27 EIVLTQSPGTLSLSPGERATLSCSVSSSVSYMHWYQQKPGQAPKLLIYDTSKLASGIPDRFSGSGSGTDYTLTISRLEPED FAVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3212 VL D-H.28 DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQKPGKAPKSLIYDTSKLASGVPSKFSGSGSGTDYTLTISSLQPED FATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3213 VL D-H.29 DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQKPEKAPKSLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3214 VL D-H.30 DIVLTQSPDSLAVSLGERATINCSVSSSVSYMHWYQQKPGQPPKLLIYDTSKLASGVPDRFSGSGSGTDYTLTISSLQAE DVAVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3215 VL D-H.31 EIVLTQTPLSLSITPGEQASMSCSVSSSVSYMHWYLQKARPVPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISRVEAE DFGVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3216 VL D-H.32 EIVLTQTPLSLSITPGEQASISCSVSSSVSYMHWYLQKARPVPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISRVEAED FGVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3217 VL D-H.33 DIVLTQSPLSLPVTPGEPASISCSVSSSVSYMHWYLQKPGQSPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISRVEAE DVGVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3218 VL D-H.34 DIVLTQSPLSLPVTLGQPASISCSVSSSVSYMHWYQQRPGQSPKRLIYDTSKLASGVPDRFSGSGSGTDYTLKISRVEAE DVGVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3219 VL D-H.35 DIVLTQTPLSLPVTPGEPASISCSVSSSVSYMHWYLQKPGQSPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISRVEAE DVGVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3220 VL D-H.36 DIVLTQTPLSLSVTPGQPASISCSVSSSVSYMHWYLQKPGQSPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISRVEAE DVGVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3221 VL D-H.37 DIVLTQTPLSLSVTPGQPASISCSVSSSVSYMHWYLQKPGQPPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISRVEAE DVGVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3222 VL D-H.38 DIQLIQSPSFLSASVGDRVSIICSVSSSVSYMHWYLQKPGKSPKLFIYDTSKLASGVSSRFSGRGSGTDYTLTIISLKPEDFA AYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3223 VL D-H.39 DIVLTQTPLSSPVTLGQPASISCSVSSSVSYMHWYQQRPGQPPKLLIYDTSKLASGVPDRFSGSGAGTDYTLKISRVEAE DVGVYYCQQWSSNPQYTFGQGTKLEIKSEQ ID NO: 3224 VL D-H.40 EITLTQSPAFMSATPGDKVNISCSVSSSVSYMHWYQQKPGEAPKFIIYDTSKLASGIPPRFSGSGYGTDYTLTINNIESED AAYYYCQQWSSNPQYTFGQGTKLEIKVariable HEAVY chain (VH) SEQ ID NO: 3225 VH D-H.1EVQLVESGGGLVKPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLKTEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3226 VH D-H.2EVQLVESGGALVKPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLKTEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3227 VH D-H.3EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3228 VH D-H.4EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3229 VH D-H.5EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNSLYLQMNSLKTEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3230 VH D-H.6EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNAKNSLYLQMNSLRAEDMAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3231 VH D-H.7EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGQFTISRDNAKNTLYLQMNSLRAEDMAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3232 VH D-H.8EVQLVESGGGLVKPGRSLRLSCTVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNILYLQMNSLKTEDTAVYYCSRHGGNFFDYWGQGTTVTVSS SEQ ID NO: 3233 VH D-H.9EVQLVESGGGLVKPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3234 VH D-H.10EVQLVESGGGLVQPGGSLKLSCAVSGFTFRSYGMSWVRQASGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLKTEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3235 VH D-H.11QVQLVESGGGVVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3236 VH D-H.12QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3237 VH D-H.13EVQLVESGGGLVQPGGSLRLSCPVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNANNSLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTVS S SEQ ID NO: 3238 VH D-H.14EVQLVESGGGLVQPGRSLRLSCTVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNILYLQMNSLKTEDTAVYYCSRHGGNFFDYWGQGTTVTVSS SEQ ID NO: 3239 VH D-H.15EVQLVESGGGLVQPGPSLRLSCTVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNILYLQMNSLKTEDTAVYYCSRHGGNFFDYWGQGTTVTVSS SEQ ID NO: 3240 VH D-H.16EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3241 VH D-H.17EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3242 VH D-H.18QVQLVESGGGLVKPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3243 VH D-H.19QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3244 VH D-H.20EVQLLESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTVS S SEQ ID NO: 3245 VH D-H.21EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRHNSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3246 VH D-H.22EVQLVESGGGLIQPGGSLRLSCAVSGFTFRSYGMSWVRQPPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTVSS SEQ ID NO: 3247 VH D-H.23EVQLVESGGGLIQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTVS S SEQ ID NO: 3248 VH D-H.24EVQLVESGGGLVQPGRSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3249 VH D-H.25QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3250 VH D-H.26QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEGTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3251 VH D-H.27QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFAISRDNSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3252 VH D-H.28QVQLVDSGGGVVQPGRSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3253 VH D-H.29EVQLVESGGGVVRPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3254 VH D-H.30EVQLVESGGVVVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNSLYLQMNSLRAEDTALYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3255 VH D-H.31EVQLVESGGGVVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3256 VH D-H.32EVQLVESGGVVVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3257 VH D-H.33EVQLVETGGGLIQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTVS S SEQ ID NO: 3258 VH D-H.34EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQATGKGLEWVALISSGGSYTYYTDSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3259 VH D-H.35EVQLVESRGVLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3260 VH D-H.36EVQLVESGGGLVQPGRSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNAKNSLYLQMNSLRAEDMALYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3261 VH D-H.37QVQLVESGGGLVQPGGSLRLSCSVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3262 VH D-H.38EVQLVESGGGLVQPGGSLRLSCSVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTVS S SEQ ID NO: 3263 VH D-H.39QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSTNTLFLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3264 VH D-H.40QVQLLESGGGLVKPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3265 VH D-H.41EVQLVESGEGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMGSLRAEDMAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3266 VH D-H.42EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMGSLRAEDMAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3267 VH D-H.43EVQLVESGGGLVQPGGSLRLSCSVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYVQMSSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTVS S SEQ ID NO: 3268 VH D-H.44EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFIISRDNSRNSLYLQKNRRRAEDMAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3269 VH D-H.45EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWVHQAPGKGLEWVALISSGGSYTYYTDSVKGRFIISRDNSRNTLYLQTNSLRAEDTAVYYCSRHGGNFFDYWGQGTTVTVS S SEQ ID NO: 3270 VH D-H.46EVHLVESGGGLVQPGGALRLSCAVSGFTFRSYGMSWVRQATGKGLEWVALISSGGSYTYYTDSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3271 VH D-H.47EVQLVESGGGLVQPRGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNNLRAEGTAVYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3272 VH D-H.48EVQLVESGGGLVQPRGSLRLSCAVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNSKNTLYLQMNNLRAEGTAAYYCSRHGGNFFDYWGQGTTVT VSS SEQ ID NO: 3273 VH D-H.49QVQLVQSGAEVKKPGASVKVSCKVSGFTFRSYGMSWVRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTITRDNSTNTLYMELSSLRSEDTAVYYCSRHGGNFFDYWGQGTTVTV SS SEQ ID NO: 3274 VH D-H.50QVQLVQSGSELKKPGASVKVSCKVSGFTFRSYGMSWVRQAPGQGLEWVALISSGGSYTYYTDSVKGRFVISRDNSVNTLYLQISSLKAEDTAVYYCSRHGGNFFDYWGQGTTVTVS S

In some embodiments, the anti-TCRβ V10 antibody molecule comprises a VHor a VL of an antibody described in Table 12, or a sequence with atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβ V10 antibody molecule comprises a VHand a VL of an antibody described in Table 12, or a sequence with atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

Antibody Molecules

In one embodiment, the antibody molecule binds to a cancer antigen,e.g., a tumor antigen or a stromal antigen. In some embodiments, thecancer antigen is, e.g., a mammalian, e.g., a human, cancer antigen. Inother embodiments, the antibody molecule binds to an immune cellantigen, e.g., a mammalian, e.g., a human, immune cell antigen. Forexample, the antibody molecule binds specifically to an epitope, e.g.,linear or conformational epitope, on the cancer antigen or the immunecell antigen.

In an embodiment, an antibody molecule is a monospecific antibodymolecule and binds a single epitope. E.g., a monospecific antibodymolecule having a plurality of immunoglobulin variable domain sequences,each of which binds the same epitope.

In an embodiment an antibody molecule is a multispecific ormultifunctional antibody molecule, e.g., it comprises a plurality ofimmunoglobulin variable domains sequences, wherein a firstimmunoglobulin variable domain sequence of the plurality has bindingspecificity for a first epitope and a second immunoglobulin variabledomain sequence of the plurality has binding specificity for a secondepitope. In an embodiment the first and second epitopes are on the sameantigen, e.g., the same protein (or subunit of a multimeric protein). Inan embodiment the first and second epitopes overlap. In an embodimentthe first and second epitopes do not overlap. In an embodiment the firstand second epitopes are on different antigens, e.g., the differentproteins (or different subunits of a multimeric protein). In anembodiment a multispecific antibody molecule comprises a third, fourthor fifth immunoglobulin variable domain. In an embodiment, amultispecific antibody molecule is a bispecific antibody molecule, atrispecific antibody molecule, or a tetraspecific antibody molecule.

In an embodiment a multispecific antibody molecule is a bispecificantibody molecule. A bispecific antibody has specificity for no morethan two antigens. A bispecific antibody molecule is characterized by afirst immunoglobulin variable domain sequence which has bindingspecificity for a first epitope and a second immunoglobulin variabledomain sequence that has binding specificity for a second epitope. In anembodiment the first and second epitopes are on the same antigen, e.g.,the same protein (or subunit of a multimeric protein). In an embodimentthe first and second epitopes overlap. In an embodiment the first andsecond epitopes do not overlap. In an embodiment the first and secondepitopes are on different antigens, e.g., the different proteins (ordifferent subunits of a multimeric protein). In an embodiment abispecific antibody molecule comprises a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a first epitope and a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a second epitope. In an embodiment a bispecific antibodymolecule comprises a half antibody having binding specificity for afirst epitope and a half antibody having binding specificity for asecond epitope. In an embodiment a bispecific antibody moleculecomprises a half antibody, or fragment thereof, having bindingspecificity for a first epitope and a half antibody, or fragmentthereof, having binding specificity for a second epitope. In anembodiment a bispecific antibody molecule comprises a scFv or a Fab, orfragment thereof, have binding specificity for a first epitope and ascFv or a Fab, or fragment thereof, have binding specificity for asecond epitope.

In an embodiment, an antibody molecule comprises a diabody, and asingle-chain molecule, as well as an antigen-binding fragment of anantibody (e.g., Fab, F(ab′)₂, and Fv). For example, an antibody moleculecan include a heavy (H) chain variable domain sequence (abbreviatedherein as VH), and a light (L) chain variable domain sequence(abbreviated herein as VL). In an embodiment an antibody moleculecomprises or consists of a heavy chain and a light chain (referred toherein as a half antibody. In another example, an antibody moleculeincludes two heavy (H) chain variable domain sequences and two light (L)chain variable domain sequence, thereby forming two antigen bindingsites, such as Fab, Fab′, F(ab′)₂, Fc, Fd, Fd′, Fv, single chainantibodies (scFv for example), single variable domain antibodies,diabodies (Dab) (bivalent and bispecific), and chimeric (e.g.,humanized) antibodies, which may be produced by the modification ofwhole antibodies or those synthesized de novo using recombinant DNAtechnologies. These functional antibody fragments retain the ability toselectively bind with their respective antigen or receptor. Antibodiesand antibody fragments can be from any class of antibodies including,but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass(e.g., IgG1, IgG2, IgG3, and IgG4) of antibodies. The a preparation ofantibody molecules can be monoclonal or polyclonal. An antibody moleculecan also be a human, humanized, CDR-grafted, or in vitro generatedantibody. The antibody can have a heavy chain constant region chosenfrom, e.g., IgG1, IgG2, IgG3, or IgG4. The antibody can also have alight chain chosen from, e.g., kappa or lambda. The term“immunoglobulin” (Ig) is used interchangeably with the term “antibody”herein.

Examples of antigen-binding fragments of an antibody molecule include:(i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region;(iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fvfragment consisting of the VL and VH domains of a single arm of anantibody, (v) a diabody (dAb) fragment, which consists of a VH domain;(vi) a camelid or camelized variable domain; (vii) a single chain Fv(scFv), see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) a singledomain antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

Antibody molecules include intact molecules as well as functionalfragments thereof. Constant regions of the antibody molecules can bealtered, e.g., mutated, to modify the properties of the antibody (e.g.,to increase or decrease one or more of: Fc receptor binding, antibodyglycosylation, the number of cysteine residues, effector cell function,or complement function).

Antibody molecules can also be single domain antibodies. Single domainantibodies can include antibodies whose complementary determiningregions are part of a single domain polypeptide. Examples include, butare not limited to, heavy chain antibodies, antibodies naturally devoidof light chains, single domain antibodies derived from conventional4-chain antibodies, engineered antibodies and single domain scaffoldsother than those derived from antibodies. Single domain antibodies maybe any of the art, or any future single domain antibodies. Single domainantibodies may be derived from any species including, but not limited tomouse, human, camel, llama, fish, shark, goat, rabbit, and bovine.According to another aspect of the invention, a single domain antibodyis a naturally occurring single domain antibody known as heavy chainantibody devoid of light chains. Such single domain antibodies aredisclosed in WO 9404678, for example. For clarity reasons, this variabledomain derived from a heavy chain antibody naturally devoid of lightchain is known herein as a VHH or nanobody to distinguish it from theconventional VH of four chain immunoglobulins. Such a VHH molecule canbe derived from antibodies raised in Camelidae species, for example incamel, llama, dromedary, alpaca and guanaco. Other species besidesCamelidae may produce heavy chain antibodies naturally devoid of lightchain; such VHHs are within the scope of the invention.

The VH and VL regions can be subdivided into regions ofhypervariability, termed “complementarity determining regions” (CDR),interspersed with regions that are more conserved, termed “frameworkregions” (FR or FW).

The extent of the framework region and CDRs has been precisely definedby a number of methods (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Chothia, C. etal. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used byOxford Molecular's AbM antibody modeling software. See, generally, e.g.,Protein Sequence and Structure Analysis of Antibody Variable Domains.In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R.,Springer-Verlag, Heidelberg).

The terms “complementarity determining region,” and “CDR,” as usedherein refer to the sequences of amino acids within antibody variableregions which confer antigen specificity and binding affinity. Ingeneral, there are three CDRs in each heavy chain variable region(HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region(LCDR1, LCDR2, LCDR3).

The precise amino acid sequence boundaries of a given CDR can bedetermined using any of a number of known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme).As used herein, the CDRs defined according the “Chothia” number schemeare also sometimes referred to as “hypervariable loops.”

For example, under Kabat, the CDR amino acid residues in the heavy chainvariable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and95-102 (HCDR3); and the CDR amino acid residues in the light chainvariable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and89-97 (LCDR3). Under Chothia, the CDR amino acids in the VH are numbered26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acidresidues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96(LCDR3).

Each VH and VL typically includes three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

The antibody molecule can be a polyclonal or a monoclonal antibody.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope. Amonoclonal antibody can be made by hybridoma technology or by methodsthat do not use hybridoma technology (e.g., recombinant methods).

The antibody can be recombinantly produced, e.g., produced by phagedisplay or by combinatorial methods, or by yeast display.

Phage display and combinatorial methods for generating antibodies areknown in the art (as described in, e.g., Ladner et al. U.S. Pat. No.5,223,409; Kang et al. International Publication No. WO 92/18619; Doweret al. International Publication No. WO 91/17271; Winter et al.International Publication WO 92/20791; Markland et al. InternationalPublication No. WO 92/15679; Breitling et al. International PublicationWO 93/01288; McCafferty et al. International Publication No. WO92/01047; Garrard et al. International Publication No. WO 92/09690;Ladner et al. International Publication No. WO 90/02809; Fuchs et al.(1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum AntibodHybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffthset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982, the contents of all of which areincorporated by reference herein).

The yeast display method for generating or identifying antibodies isknown in the art, e.g., as described in Chao et al. (2006) NatureProtocols 1(2):755-68, the entire contents of which is incorporated byreference herein.

In one embodiment, the antibody is a fully human antibody (e.g., anantibody made in a mouse which has been genetically engineered toproduce an antibody from a human immunoglobulin sequence), or anon-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g.,monkey), camel antibody. Preferably, the non-human antibody is a rodent(mouse or rat antibody). Methods of producing rodent antibodies areknown in the art.

Human monoclonal antibodies can be generated using transgenic micecarrying the human immunoglobulin genes rather than the mouse system.Splenocytes from these transgenic mice immunized with the antigen ofinterest are used to produce hybridomas that secrete human mAbs withspecific affinities for epitopes from a human protein (see, e.g., Woodet al. International Application WO 91/00906, Kucherlapati et al. PCTpublication WO 91/10741; Lonberg et al. International Application WO92/03918; Kay et al. International Application 92/03917; Lonberg, N. etal. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet.7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon etal. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol21:1323-1326).

An antibody molecule can be one in which the variable region, or aportion thereof, e.g., the CDRs, are generated in a non-human organism,e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodiesare within the invention. Antibody molecules generated in a non-humanorganism, e.g., a rat or mouse, and then modified, e.g., in the variableframework or constant region, to decrease antigenicity in a human arewithin the invention.

An “effectively human” protein is a protein that does substantially notevoke a neutralizing antibody response, e.g., the human anti-murineantibody (HAMA) response. HAMA can be problematic in a number ofcircumstances, e.g., if the antibody molecule is administeredrepeatedly, e.g., in treatment of a chronic or recurrent diseasecondition. A HAMA response can make repeated antibody administrationpotentially ineffective because of an increased antibody clearance fromthe serum (see, e.g., Saleh et al., Cancer Immunol. Immunother.,32:180-190 (1990)) and also because of potential allergic reactions(see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

Chimeric antibodies can be produced by recombinant DNA techniques knownin the art (see Robinson et al., International Patent PublicationPCT/US86/02269; Akira, et al., European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., InternationalApplication WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabillyet al., European Patent Application 125,023; Better et al. (1988 Science240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987,J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimuraet al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

A humanized or CDR-grafted antibody will have at least one or two butgenerally all three recipient CDRs (of heavy and or light immuoglobulinchains) replaced with a donor CDR. The antibody may be replaced with atleast a portion of a non-human CDR or only some of the CDRs may bereplaced with non-human CDRs. It is only necessary to replace the numberof CDRs required for binding to the antigen. Preferably, the donor willbe a rodent antibody, e.g., a rat or mouse antibody, and the recipientwill be a human framework or a human consensus framework. Typically, theimmunoglobulin providing the CDRs is called the “donor” and theimmunoglobulin providing the framework is called the “acceptor.” In oneembodiment, the donor immunoglobulin is a non-human (e.g., rodent). Theacceptor framework is a naturally-occurring (e.g., a human) framework ora consensus framework, or a sequence about 85% or higher, preferably90%, 95%, 99% or higher identical thereto.

As used herein, the term “consensus sequence” refers to the sequenceformed from the most frequently occurring amino acids (or nucleotides)in a family of related sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofproteins, each position in the consensus sequence is occupied by theamino acid occurring most frequently at that position in the family. Iftwo amino acids occur equally frequently, either can be included in theconsensus sequence. A “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence.

An antibody molecule can be humanized by methods known in the art (seee.g., Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986,BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089,5,693,761 and 5,693,762, the contents of all of which are herebyincorporated by reference).

Humanized or CDR-grafted antibody molecules can be produced byCDR-grafting or CDR substitution, wherein one, two, or all CDRs of animmunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539;Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S.Pat. No. 5,225,539, the contents of all of which are hereby expresslyincorporated by reference. Winter describes a CDR-grafting method whichmay be used to prepare the humanized antibodies of the present invention(UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S.Pat. No. 5,225,539), the contents of which is expressly incorporated byreference.

Also within the scope of the invention are humanized antibody moleculesin which specific amino acids have been substituted, deleted or added.Criteria for selecting amino acids from the donor are described in U.S.Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089,e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of whichare hereby incorporated by reference. Other techniques for humanizingantibodies are described in Padlan et al. EP 519596 A1, published onDec. 23, 1992.

The antibody molecule can be a single chain antibody. A single-chainantibody (scFV) may be engineered (see, for example, Colcher, D. et al.(1999) Ann NY Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res2:245-52). The single chain antibody can be dimerized or multimerized togenerate multivalent antibodies having specificities for differentepitopes of the same target protein.

In yet other embodiments, the antibody molecule has a heavy chainconstant region chosen from, e.g., the heavy chain constant regions ofIgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly,chosen from, e.g., the (e.g., human) heavy chain constant regions ofIgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody moleculehas a light chain constant region chosen from, e.g., the (e.g., human)light chain constant regions of kappa or lambda. The constant region canbe altered, e.g., mutated, to modify the properties of the antibody(e.g., to increase or decrease one or more of: Fc receptor binding,antibody glycosylation, the number of cysteine residues, effector cellfunction, and/or complement function). In one embodiment the antibodyhas: effector function; and can fix complement. In other embodiments theantibody does not; recruit effector cells; or fix complement. In anotherembodiment, the antibody has reduced or no ability to bind an Fcreceptor. For example, it is a isotype or subtype, fragment or othermutant, which does not support binding to an Fc receptor, e.g., it has amutagenized or deleted Fc receptor binding region.

Methods for altering an antibody constant region are known in the art.Antibodies with altered function, e.g. altered affinity for an effectorligand, such as FcR on a cell, or the Cl component of complement can beproduced by replacing at least one amino acid residue in the constantportion of the antibody with a different residue (see e.g., EP 388,151A1, U.S. Pat. Nos. 5,624,821 and 5,648,260, the contents of all of whichare hereby incorporated by reference). Similar type of alterations couldbe described which if applied to the murine, or other speciesimmunoglobulin would reduce or eliminate these functions.

An antibody molecule can be derivatized or linked to another functionalmolecule (e.g., another peptide or protein). As used herein, a“derivatized” antibody molecule is one that has been modified. Methodsof derivatization include but are not limited to the addition of afluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinityligand such as biotin. Accordingly, the antibody molecules of theinvention are intended to include derivatized and otherwise modifiedforms of the antibodies described herein, including immunoadhesionmolecules. For example, an antibody molecule can be functionally linked(by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as anotherantibody (e.g., a bispecific antibody or a diabody), a detectable agent,a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptidethat can mediate association of the antibody or antibody portion withanother molecule (such as a streptavidin core region or a polyhistidinetag).

One type of derivatized antibody molecule is produced by crosslinkingtwo or more antibodies (of the same type or of different types, e.g., tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Multispecific or Multifunctional Antibody Molecules

Exemplary structures of multispecific and multifunctional moleculesdefined herein are described throughout. Exemplary structures arefurther described in: Weidle U et al. (2013) The Intriguing Options ofMultispecific Antibody Formats for Treatment of Cancer. Cancer Genomics& Proteomics 10: 1-18 (2013); and Spiess C et al. (2015) Alternativemolecular formats and therapeutic applications for bispecificantibodies. Molecular Immunology 67: 95-106; the full contents of eachof which is incorporated by reference herein).

In embodiments, multispecific antibody molecules can comprise more thanone antigen-binding site, where different sites are specific fordifferent antigens. In embodiments, multispecific antibody molecules canbind more than one (e.g., two or more) epitopes on the same antigen. Inembodiments, multispecific antibody molecules comprise anantigen-binding site specific for a target cell (e.g., cancer cell) anda different antigen-binding site specific for an immune effector cell.In one embodiment, the multispecific antibody molecule is a bispecificantibody molecule. Bispecific antibody molecules can be classified intofive different structural groups: (i) bispecific immunoglobulin G(BsIgG); (ii) IgG appended with an additional antigen-binding moiety;(iii) bispecific antibody fragments; (iv) bispecific fusion proteins;and (v) bispecific antibody conjugates.

BsIgG is a format that is monovalent for each antigen. Exemplary BsIgGformats include but are not limited to crossMab, DAF (two-in-one), DAF(four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holesassembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab,κλ-body, orthogonal Fab. See Spiess et al. Mol. Immunol.67(2015):95-106. Exemplary BslgGs include catumaxomab (FreseniusBiotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and ananti-EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech),which targets CD3 and HER2. In some embodiments, BsIgG comprises heavychains that are engineered for heterodimerization. For example, heavychains can be engineered for heterodimerization using a“knobs-into-holes” strategy, a SEED platform, a common heavy chain(e.g., in κλ-bodies), and use of heterodimeric Fc regions. See Spiess etal. Mol. Immunol. 67(2015):95-106. Strategies that have been used toavoid heavy chain pairing of homodimers in BsIgG include knobs-in-holes,duobody, azymetric, charge pair, HA-TF, SEEDbody, and differentialprotein A affinity. See Id. BsIgG can be produced by separate expressionof the component antibodies in different host cells and subsequentpurification/assembly into a BsIgG. BsIgG can also be produced byexpression of the component antibodies in a single host cell. BsIgG canbe purified using affinity chromatography, e.g., using protein A andsequential pH elution.

IgG appended with an additional antigen-binding moiety is another formatof bispecific antibody molecules. For example, monospecific IgG can beengineered to have bispecificity by appending an additionalantigen-binding unit onto the monospecific IgG, e.g., at the N- orC-terminus of either the heavy or light chain. Exemplary additionalantigen-binding units include single domain antibodies (e.g., variableheavy chain or variable light chain), engineered protein scaffolds, andpaired antibody variable domains (e.g., single chain variable fragmentsor variable fragments). See Id. Examples of appended IgG formats includedual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG,IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V,V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, andDVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106.An example of an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), whichbinds IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie),which binds IL-1α and IL-1β; and ABT-122 (AbbVie), which binds TNF andIL-17A.

Bispecific antibody fragments (BsAb) are a format of bispecific antibodymolecules that lack some or all of the antibody constant domains. Forexample, some BsAb lack an Fc region. In embodiments, bispecificantibody fragments include heavy and light chain regions that areconnected by a peptide linker that permits efficient expression of theBsAb in a single host cell. Exemplary bispecific antibody fragmentsinclude but are not limited to nanobody, nanobody-HAS, BiTE, Diabody,DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body,miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv,scFv-CH-CL-scFv, F(ab′) 2, F(ab′) 2-scFv2, scFv-KIH, Fab-scFv-Fc,tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, andintrabody. See Id. For example, the BiTE format comprises tandem scFvs,where the component scFvs bind to CD3 on T cells and a surface antigenon cancer cells

Bispecific fusion proteins include antibody fragments linked to otherproteins, e.g., to add additional specificity and/or functionality. Anexample of a bispecific fusion protein is an immTAC, which comprises ananti-CD3 scFv linked to an affinity-matured T-cell receptor thatrecognizes HLA-presented peptides. In embodiments, the dock-and-lock(DNL) method can be used to generate bispecific antibody molecules withhigher valency. Also, fusions to albumin binding proteins or human serumalbumin can be extend the serum half-life of antibody fragments. See Id.

In embodiments, chemical conjugation, e.g., chemical conjugation ofantibodies and/or antibody fragments, can be used to create BsAbmolecules. See Id. An exemplary bispecific antibody conjugate includesthe CovX-body format, in which a low molecular weight drug is conjugatedsite-specifically to a single reactive lysine in each Fab arm or anantibody or fragment thereof. In embodiments, the conjugation improvesthe serum half-life of the low molecular weight drug. An exemplaryCovX-body is CVX-241 (NCT01004822), which comprises an antibodyconjugated to two short peptides inhibiting either VEGF or Ang2. See Id.

The antibody molecules can be produced by recombinant expression, e.g.,of at least one or more component, in a host system. Exemplary hostsystems include eukaryotic cells (e.g., mammalian cells, e.g., CHOcells, or insect cells, e.g., SF9 or S2 cells) and prokaryotic cells(e.g., E. coli). Bispecific antibody molecules can be produced byseparate expression of the components in different host cells andsubsequent purification/assembly. Alternatively, the antibody moleculescan be produced by expression of the components in a single host cell.Purification of bispecific antibody molecules can be performed byvarious methods such as affinity chromatography, e.g., using protein Aand sequential pH elution. In other embodiments, affinity tags can beused for purification, e.g., histidine-containing tag, myc tag, orstreptavidin tag.

Exemplary Bispecific Molecules

In an aspect, a multispecific molecule disclosed herein comprises asequence disclosed herein, e.g., a sequence chosen from SEQ ID NOs:1004-1007, 3275-3277, 3286, or 3287, or a sequence with at least 85%,90%, 955, 96%, 97%, 98%, 99% or more identity thereto. In someembodiments, a multispecific molecule disclosed herein comprises aleader sequence comprising the amino acid sequence of SEQ ID NO: 3288.In some embodiments, a multispecific molecule disclosed herein does notcomprise a leader sequence comprising the amino acid sequence of SEQ IDNO: 3288.

Molecule F: aCD19×aVb6.5

Molecule F comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 1004 and a light chain comprising the amino acid sequence ofSEQ ID NO: 1005.

Molecule F.1

(heavy chain) (Tcrvbeta6_5 scFv/anti-CD19 heavy chain) SEQ ID NO: 1004METDTLLLWVLLLWVPGSTGQVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVGINVVWHQQKPGKAPKALIYSSSHRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIKGGGGSQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVFLTMTNMDPVDTATYYCARMELWSYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Molecule F.2

(light chain) (anti-CD19 light chain) SEQ ID NO: 1005METPAQLLFLLLLWLPDTTGENVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDHTLTISSLEPEDFAVYYCFQGSVYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In an aspect, a multispecific molecule disclosed herein comprises SEQ IDNO: 1004 and/or SEQ ID NO: 1005 or a sequence with at least 85%, 90%,955, 96%, 97%, 98%, 99% or more identity thereto.

Molecule G: aBCMA×aVb6.5

Molecule G comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 1006 and a light chain comprising the amino acid sequence ofSEQ ID NO: 1007.

Molecule G.1

(heavy chain) SEQ ID NO: 1006METDTLLLWVLLLWVPGSTGQVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVGINVVWHQQKPGKAPKALIYSSSHRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIKGGGGSQVQLVESGGGVVQPGRSLRLSCAASGIDFSRYWMSWVRQAPGKGLEWVGEINPDSSTINYAPSLKDRFTISRDNSKNTLYLQMSSLRAEDTAVYYCASLYYDYGDAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

Molecule G.2

(light chain) SEQ ID NO: 1007METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCKASQSVDSNVAWYQQKPEKAPKALIFSASLRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNNYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In an aspect, a multispecific molecule disclosed herein comprises SEQ IDNO: 1006 and/or SEQ ID NO: 1007 or a sequence with at least 85%, 90%,955, 96%, 97%, 98%, 99% or more identity thereto.

Molecule H: aBCMA×aTCRvbeta6_5

Molecule H comprises a first heavy chain comprising the amino acidsequence of SEQ ID NO: 3275, a light chain comprising the amino acidsequence of SEQ ID NO: 3277, and a second heavy chain comprising theamino acid sequence of SEQ ID NO: 3276.

Molecule H.1

(anti-BCMA heavy chain) SEQ ID NO: 3275METDTLLLWVLLLWVPGSTGQVQLVESGGGVVQPGRSLRLSCAASGIDFSRYWMSWVRQAPGKGLEWVGEINPDSSTINYAPSLKDRFTISRDNSKNTLYLQMSSLRAEDTAVYYCASLYYDYGDAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Molecule H.2

(TCRvbeta_6_5 scFv humanized) SEQ ID NO: 3276METDTLLLWVLLLWVPGSTGQVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCAGSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQNVGINVVWHQQKPGKAPKALIYSSSHRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGTKLEIKGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Molecule H.3

(anti-BCMA light chain) SEQ ID NO: 3277METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCKASQSVDSNVAWYQQKPEKAPKALIFSASLRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNNYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In an aspect, a multispecific molecule disclosed herein comprises SEQ IDNO: 3275, SEQ ID NO: 3276, and/or SEQ ID NO: 3277 or a sequence with atleast 85%, 90%, 955, 96%, 97%, 98%, 99% or more identity thereto.

Molecule I: Half Arm BCMA Fab with c-Terminal scFv TCRvbeta

Molecule I comprises a first heavy chain comprising the amino acidsequence of SEQ ID NO: 3286, a light chain comprising the amino acidsequence of SEQ ID NO: 3277, and a second heavy chain comprising theamino acid sequence of SEQ ID NO: 3287.

Molecule I.1

(heavy chain 1) SEQ ID NO: 3286METDTLLLWVLLLWVPGSTGQVQLVESGGGVVQPGRSLRLSCAASGIDFSRYWMSWVRQAPGKGLEWVGEINPDSSTINYAPSLKDRFTISRDNSKNTLYLQMSSLRAEDTAVYYCASLYYDYGDAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL

Molecule I.2

(light chain) SEQ ID NO: 3277METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCKASQSVDSNVAWYQQKPEKAPKALIFSASLRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNNYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Molecule I.3

(heavy chain 2) SEQ ID NO: 3287METDTLLLWVLLLWVPGSTGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK

In an aspect, a multispecific molecule disclosed herein comprises SEQ IDNO: 3286, SEQ ID NO: 3277, and/or SEQ ID NO: 3287 or a sequence with atleast 85%, 90%, 955, 96%, 97%, 98%, 99% or more identity thereto.

Antibody-Like Frameworks or Scaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed in the anti-TCRvb antibody molecules disclosed herein ormultifunctional formats thereof so long as the resulting polypeptideincludes at least one binding region which specifically binds to thetarget antigen, e.g., a TCRvb, a tumor antigen, among others. Suchframeworks or scaffolds include the 5 main idiotypes of humanimmunoglobulins, or fragments thereof, and include immunoglobulins ofother animal species, preferably having humanized aspects. Novelframeworks, scaffolds and fragments continue to be discovered anddeveloped by those skilled in the art.

In one embodiment, the anti-TCRvb antibody molecules disclosed herein ormultifunctional formats thereof include non-immunoglobulin basedantibodies using non-immunoglobulin scaffolds onto which CDRs can begrafted. Any non-immunoglobulin frameworks and scaffolds may beemployed, as long as they comprise a binding region specific for thetarget antigen (e.g., TCRvb or a tumor antigen). Exemplarynon-immunoglobulin frameworks or scaffolds include, but are not limitedto, fibronectin (Compound Therapeutics, Inc., Waltham, Mass.), ankyrin(Molecular Partners AG, Zurich, Switzerland), domain antibodies(Domantis, Ltd., Cambridge, Mass., and Ablynx nv, Zwijnaarde, Belgium),lipocalin (Pieris Proteolab AG, Freising, Germany), small modularimmuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Wash.),maxybodies (Avidia, Inc., Mountain View, Calif.), Protein A (AffibodyAG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil ProteinsGmbH, Halle, Germany).

Fibronectin scaffolds are typically based on fibronectin type III domain(e.g., the tenth module of the fibronectin type III (10 Fn3 domain)).The fibronectin type III domain has 7 or 8 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (see U.S. Pat. No.6,818,418). Because of this structure, the non-immunoglobulin antibodymimics antigen binding properties that are similar in nature andaffinity to those of antibodies. These scaffolds can be used in a looprandomization and shuffling strategy in vitro that is similar to theprocess of affinity maturation of antibodies in vivo. Thesefibronectin-based molecules can be used as scaffolds where the loopregions of the molecule can be replaced with CDRs of the invention usingstandard cloning techniques.

The ankyrin technology is based on using proteins with ankyrin derivedrepeat modules as scaffolds for bearing variable regions which can beused for binding to different targets. The ankyrin repeat moduletypically is a about 33 amino acid polypeptide consisting of twoanti-parallel α-helices and a β-turn. Binding of the variable regionscan be optimized by using ribosome display.

Avimers are used by nature for protein-protein interactions and in humanover 250 proteins are structurally based on A-domains. Avimers consistof a number of different “A-domain” monomers (2-10) linked via aminoacid linkers. Avimers can be created that can bind to the target antigenusing the methodology described in, for example, U.S. Patent ApplicationPublication Nos. 20040175756; 20050053973; 20050048512; and 20060008844.

Affibody affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate affibody libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibodymolecules mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of affibody molecules issimilar to that of an antibody.

Anticalins are known commercially, e.g., Pieris ProteoLab AG. They arederived from lipocalins, a widespread group of small and robust proteinsthat are usually involved in the physiological transport or storage ofchemically sensitive or insoluble compounds. Several natural lipocalinsoccur in human tissues or body liquids. The protein architecture isreminiscent of immunoglobulins, with hypervariable loops on top of arigid framework. However, in contrast with antibodies or theirrecombinant fragments, lipocalins are composed of a single polypeptidechain with 160 to 180 amino acid residues, being just marginally biggerthan a single immunoglobulin domain. The set of four loops, which makesup the binding pocket, shows pronounced structural plasticity andtolerates a variety of side chains. The binding site can thus bereshaped in a proprietary process in order to recognize prescribedtarget molecules of different shape with high affinity and specificity.One protein of lipocalin family, the bilin-binding protein (BBP) ofPieris Brassicae has been used to develop anticalins by mutagenizing theset of four loops. One example of a patent application describinganticalins is in PCT Publication No. WO 199916873.

Affilin molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New affilin molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.Affilin molecules do not show any structural homology to immunoglobulinproteins. Currently, two affilin scaffolds are employed, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368.

Protein epitope mimetics (PEM) are medium-sized, cyclic, peptide-likemolecules (MW 1-2 kDa) mimicking beta-hairpin secondary structures ofproteins, the major secondary structure involved in protein-proteininteractions.

Domain antibodies (dAbs) can be used in the anti-TCRvb antibodymolecules disclosed herein or multifunctional formats thereof are smallfunctional binding fragments of antibodies, corresponding to thevariable regions of either the heavy or light chains of antibodies.Domain antibodies are well expressed in bacterial, yeast, and mammaliancell systems. Further details of domain antibodies and methods ofproduction thereof are known in the art (see, for example, U.S. Pat.Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; EuropeanPatents 0368684 & 0616640; WO05/035572, WO04/101790, WO04/081026,WO04/058821, WO04/003019 and WO03/002609. Nanobodies are derived fromthe heavy chains of an antibody.

A nanobody typically comprises a single variable domain and two constantdomains (CH2 and CH3) and retains antigen-binding capacity of theoriginal antibody. Nanobodies can be prepared by methods known in theart (See e.g., U.S. Pat. Nos. 6,765,087, 6,838,254, WO 06/079372).Unibodies consist of one light chain and one heavy chain of an IgG4antibody. Unibodies may be made by the removal of the hinge region ofIgG4 antibodies. Further details of unibodies and methods of preparingthem may be found in WO2007/059782.

Tumor Antigen Moiety

In an aspect, provided herein is a multispecific molecule, e.g., abispecific molecule, comprising:

(i) a first moiety (e.g., a first immune cell engager) comprising theanti-TCRβV antibody molecule described herein; and(ii) a second moiety comprising one or more of: a tumor-targetingmoiety; a second immune cell engager; a cytokine molecule or a stromalmodifying moiety.

In some embodiments of any of the compositions or methods disclosedherein, the tumor-targeting moiety is an antigen, e.g., a cancerantigen. In some embodiments, the cancer antigen is a tumor antigen orstromal antigen, or a hematological antigen.

In some embodiments of any of the compositions or methods disclosedherein, the tumor-targeting moiety, e.g., cancer antigen, is chosenfrom: BCMA, FcRH5, CD19, CD20, CD22, CD30, CD33, CD38, CD47, CD99,CD123, FcRH5, CLEC12, CD179A, SLAMF7, or NY-ESO1, PDL1, CD47, gangloside2 (GD2), prostate stem cell antigen (PSCA), prostate specific membraneantigen (PMSA), prostate-specific antigen (PSA), carcinoembryonicantigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72,BING-4, Calcium-activated chloride channel 2, Cyclin-B1, 9D7, Ep-CAM,EphA3, Her2/neu, Telomerase, SAP-1, Survivin, NY-ESO-1/LAGE-1, PRAME,SSX-2, Melan-A/MART-1, Gp100/pme117, Tyrosinase, TRP-1/-2, MC1R,β-catenin, BRCA1/2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor,AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, 13-catenin, CDK4,CDC27, a actinin-4, TRP1/gp75, TRP2, gp100, Melan-A/MART1, gangliosides,WT1, EphA3, Epidermal growth factor receptor (EGFR), MART-2, MART-1,MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, RCC, RUI1, RUI2,SAGE, TRG, TRP1, TSTA, Folate receptor alpha, L1-CAM, CAIX, gpA33, GD3,GM2, VEGFR, Intergrins (Integrin alphaVbeta3, Integrin alpha5Beta1),Carbohydrates (Le), IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, (FAP),TGF-beta, hyaluronic acid, collagen, e.g., collagen IV, tenascin C, ortenascin W. In some embodiments, the tumor-targeting moiety, e.g.,cancer antigen, is BCMA. In some embodiments, the tumor-targetingmoiety, e.g., cancer antigen, is FcRH5.

FcRH5 Targeting Moieties

In some embodiments, the multispecific molecules disclosed hereininclude a targeting moiety that binds to FcRH5 (e.g., a FcRH5 targetingmoiety). The FcRH5 targeting moiety can be chosen from an antibodymolecule (e.g., an antigen binding domain as described herein), areceptor or a receptor fragment, or a ligand or a ligand fragment, or acombination thereof. In some embodiments, the FcRH5 targeting moietyassociates with, e.g., binds to, a cancer or hematopoietic cell (e.g., amolecule, e.g., antigen, present on the surface of the cancer orhematopoietic cell). In certain embodiments, the FcRH5 targeting moietytargets, e.g., directs the multispecific molecules disclosed herein to acancer or hematopoietic cell. In some embodiments, the cancer is ahematological cancer, e.g., multiple myeloma.

In some embodiments, the multispecific molecule, e.g., the FcRH5targeting moiety, binds to a FcRH5 antigen on the surface of a cell,e.g., a cancer or hematopoietic cell. The FcRH5 antigen can be presenton a primary tumor cell, or a metastatic lesion thereof. In someembodiments, the cancer is a hematological cancer, e.g., multiplemyeloma. For example, the FcRH5 antigen can be present on a tumor, e.g.,a tumor of a class typified by having one or more of: limited tumorperfusion, compressed blood vessels, or fibrotic tumor interstitium.

The multispecific molecules described herein includes a FcRH5 targetingmoiety that comprises an anti-FcRH5 antibody or antigen-binding fragmentthereof described in U.S. Pat. No. 7,999,077, US20150098900, U.S. Pat.Nos. 8,299,220, 7,105,149, 8,362,213, 8,466,260, 8,617,559,US20160368985, US20150166661, and US20080247944, the entire contents ofany of the aforesaid publications are herein incorporated by reference.

In some embodiments, the multispecific molecules described hereinincludes a FcRH5 targeting moiety that comprises an anti-FcRH5 antibodyor antigen-binding fragment thereof described in U.S. Pat. No.7,999,077, the entire contents of which are herein incorporated byreference.

BCMA Targeting Moieties

In certain embodiments, the multispecific molecules disclosed hereininclude a targeting moiety that binds to BCMA (e.g., a BCMA targetingmoiety). The BCMA targeting moiety can be chosen from an antibodymolecule (e.g., an antigen binding domain as described herein), areceptor or a receptor fragment, or a ligand or a ligand fragment, or acombination thereof. In some embodiments, the BCMA targeting moietyassociates with, e.g., binds to, a cancer or hematopoietic cell (e.g., amolecule, e.g., antigen, present on the surface of the cancer orhematopoietic cell). In certain embodiments, the BCMA targeting moietytargets, e.g., directs the multispecific molecules disclosed herein to acancer or hematopoietic cell. In some embodiments, the cancer is ahematological cancer, e.g., multiple myeloma.

In some embodiments, the multispecific molecule, e.g., the BCMAtargeting moiety, binds to a BCMA antigen on the surface of a cell,e.g., a cancer or hematopoietic cell. The BCMA antigen can be present ona primary tumor cell, or a metastatic lesion thereof. In someembodiments, the cancer is a hematological cancer, e.g., multiplemyeloma. For example, the BCMA antigen can be present on a tumor, e.g.,a tumor of a class typified by having one or more of: limited tumorperfusion, compressed blood vessels, or fibrotic tumor interstitium.

Exemplary BCMA targeting moieties

The multispecific molecules described herein can include a BCMAtargeting moiety that comprises an anti-BCMA antibody or antigen-bindingfragment thereof described in U.S. Pat. Nos. 8,920,776, 9,243,058,9,340,621, 8,846,042, 7,083,785, 9,545,086, 7,276,241, 9,034,324,7,799,902, 9,387,237, 8,821,883, US861745, US20130273055, US20160176973,US20150368351, US20150376287, US20170022284, US20160015749,US20140242077, US20170037128, US20170051068, US20160368988,US20160311915, US20160131654, US20120213768, US20110177093,US20160297885, EP3137500, EP2699259, EP2982694, EP3029068, EP3023437,WO2016090327, WO2017021450, WO2016110584, WO2016118641, WO2016168149,the entire contents of which are incorporated herein by reference.

In one embodiment, the BCMA-targeting moiety includes an antibodymolecule (e.g., Fab or scFv) that binds to BCMA. In some embodiments,the antibody molecule to BCMA comprises one, two, or three CDRs from anyof the heavy chain variable domain sequences of Table 1, or a closelyrelated CDR, e.g., CDRs which have at least one amino acid alteration,but not more than two, three or four alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) from any ofthe CDR sequences of Table 14. In some embodiments, the antibodymolecule to BCMA comprises a heavy chain variable domain sequence chosenfrom any of the amino acid sequences of Table 14, or an amino acidsequence substantially identical thereto (e.g., 95% to 99.9% identicalthereto, or having at least one amino acid alteration, but not more thanfive, ten or fifteen alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions)).

Alternatively, or in combination with the heavy chain to BCMA disclosedherein, the antibody molecule to BCMA comprises one, two, or three CDRsfrom any of the light chain variable domain sequences of Table 14, or aclosely related CDR, e.g., CDRs which have at least one amino acidalteration, but not more than two, three or four alterations (e.g.,substitutions, deletions, or insertions, e.g., conservativesubstitutions) from any of the CDR sequences of Table 14. In someembodiments, the antibody molecule to BCMA comprises a light chainvariable domain sequence chosen from any of the amino acid sequences ofTable 14, or an amino acid sequence substantially identical thereto(e.g., 95% to 99.9% identical thereto, or having at least one amino acidalteration, but not more than five, ten or fifteen alterations (e.g.,substitutions, deletions, or insertions, e.g., conservativesubstitutions)).

TABLE 14 Amino acid sequences of exemplaryvariable regions of anti-BCMA antibodies. SEQ ID NO Description Sequence3439 83A10 VH EVQLLESGGGLVQPGGSLRLSCAASGFT FSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKVLGWFDYWGQGTLV TVSS 344083A10 VL EIVLTQSPGTLSLSPGERATLSCRASQS VSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQYGYPPDFTFGQGTKVEIK 3441 17A5 VHEVQLLESGGGLVQPGGSLRLSCAASGFT FSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKVAPYFAPFDYWGQG TLVTVSS 344217A5 VL EIVLTQSPGTLSLSPGERATLSCRASQS VSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQYGNPPLYTFGQGTKVEIK 3443 13A4 VHEVQLVQSGAEVKKPGESLKISCKGSGYS FTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWS SLKASDTAMYYCARNGYLGDYWGQGTLV TVSS 344413A4 VL DIVMTQSPLSLPVTPGEPASISCRSSQS LLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQAMQIPTFGQGTKVEIK 3445 J22.9-xiQVQLQQSGGGLVQPGGSLKLSCAASGID VH FSRYWMSWVRRAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNTLYLQMS KVRSEDTALYYCASLYYDYGDAMDYWGQ GTSVTVSS 3446J22.9-xi DIVMTQSQRFMTTSVGDRVSVTCKASQS VL VDSNVAWYQQKPRQSPKALIFSASLRFSGVPARFTGSGSGTDFTLTISNLQSEDLA EYFCQQYNNYPLTFGAGTKLELKR 3447 2A1 VHEVQLVESGGGLVKPGGSLRLSCAASGFT FGDYALSWFRQAPGKGLEWVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQ MNSLKTEDTAVYYCASSGYSSGWTPFDY WGQGTLVTVSS3448 2A1 VL QSVLTQPPSASGTPGQRVTISCSGSSSN IGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGSKSGSSASLAISGLQSEDE ADYYCAAWDDSLNGWVFGGGTKLTVLG

CDR-Grafted Scaffolds

In embodiments, the antibody molecule is a CDR-grafted scaffold domain.In embodiments, the scaffold domain is based on a fibronectin domain,e.g., fibronectin type III domain. The overall fold of the fibronectintype III (Fn3) domain is closely related to that of the smallestfunctional antibody fragment, the variable domain of the antibody heavychain. There are three loops at the end of Fn3; the positions of BC, DEand FG loops approximately correspond to those of CDR1, 2 and 3 of theVH domain of an antibody. Fn3 does not have disulfide bonds; andtherefore Fn3 is stable under reducing conditions, unlike antibodies andtheir fragments (see, e.g., WO 98/56915; WO 01/64942; WO 00/34784). AnFn3 domain can be modified (e.g., using CDRs or hypervariable loopsdescribed herein) or varied, e.g., to select domains that bind to anantigen/marker/cell described herein.

In embodiments, a scaffold domain, e.g., a folded domain, is based on anantibody, e.g., a “minibody” scaffold created by deleting three betastrands from a heavy chain variable domain of a monoclonal antibody(see, e.g., Tramontano et al., 1994, J Mol. Recognit. 7:9; and Martin etal., 1994, EMBO J. 13:5303-5309). The “minibody” can be used to presenttwo hypervariable loops. In embodiments, the scaffold domain is a V-likedomain (see, e.g., Coia et al. WO 99/45110) or a domain derived fromtendamistatin, which is a 74 residue, six-strand beta sheet sandwichheld together by two disulfide bonds (see, e.g., McConnell and Hoess,1995, J Mol. Biol. 250:460). For example, the loops of tendamistatin canbe modified (e.g., using CDRs or hypervariable loops) or varied, e.g.,to select domains that bind to a marker/antigen/cell described herein.Another exemplary scaffold domain is a beta-sandwich structure derivedfrom the extracellular domain of CTLA-4 (see, e.g., WO 00/60070).

Other exemplary scaffold domains include but are not limited to T-cellreceptors; MHC proteins; extracellular domains (e.g., fibronectin TypeIII repeats, EGF repeats); protease inhibitors (e.g., Kunitz domains,ecotin, BPTI, and so forth); TPR repeats; trifoil structures; zincfinger domains; DNA-binding proteins; particularly monomeric DNA bindingproteins; RNA binding proteins; enzymes, e.g., proteases (particularlyinactivated proteases), RNase; chaperones, e.g., thioredoxin, and heatshock proteins; and intracellular signaling domains (such as SH2 and SH3domains). See, e.g., US 20040009530 and U.S. Pat. No. 7,501,121,incorporated herein by reference.

In embodiments, a scaffold domain is evaluated and chosen, e.g., by oneor more of the following criteria: (1) amino acid sequence, (2)sequences of several homologous domains, (3) 3-dimensional structure,and/or (4) stability data over a range of pH, temperature, salinity,organic solvent, oxidant concentration. In embodiments, the scaffolddomain is a small, stable protein domain, e.g., a protein of less than100, 70, 50, 40 or 30 amino acids. The domain may include one or moredisulfide bonds or may chelate a metal, e.g., zinc.

Antibody-Based Fusions

A variety of formats can be generated which contain additional bindingentities attached to the N or C terminus of antibodies. These fusionswith single chain or disulfide stabilized Fvs or Fabs result in thegeneration of tetravalent molecules with bivalent binding specificityfor each antigen. Combinations of scFvs and scFabs with IgGs enable theproduction of molecules which can recognize three or more differentantigens.

Antibody-Fab Fusion

Antibody-Fab fusions are bispecific antibodies comprising a traditionalantibody to a first target and a Fab to a second target fused to the Cterminus of the antibody heavy chain. Commonly the antibody and the Fabwill have a common light chain. Antibody fusions can be produced by (1)engineering the DNA sequence of the target fusion, and (2) transfectingthe target DNA into a suitable host cell to express the fusion protein.It seems like the antibody-scFv fusion may be linked by a (Gly)-Serlinker between the C-terminus of the CH3 domain and the N-terminus ofthe scFv, as described by Coloma, J. et al. (1997) Nature Biotech15:159.

Antibody-scFv Fusion

Antibody-scFv Fusions are bispecific antibodies comprising a traditionalantibody and a scFv of unique specificity fused to the C terminus of theantibody heavy chain. The scFv can be fused to the C terminus throughthe Heavy Chain of the scFv either directly or through a linker peptide.Antibody fusions can be produced by (1) engineering the DNA sequence ofthe target fusion, and (2) transfecting the target DNA into a suitablehost cell to express the fusion protein. It seems like the antibody-scFvfusion may be linked by a (Gly)-Ser linker between the C-terminus of theCH3 domain and the N-terminus of the scFv, as described by Coloma, J. etal. (1997) Nature Biotech 15:159.

Variable Domain Immunoglobulin DVD

A related format is the dual variable domain immunoglobulin (DVD), whichare composed of VH and VL domains of a second specificity place upon theN termini of the V domains by shorter linker sequences.

Other exemplary multispecific antibody formats include, e.g., thosedescribed in the following US20160114057A1, US20130243775A1,US20140051833, US20130022601, US20150017187A1, US20120201746A1,US20150133638A1, US20130266568A1, US20160145340A1, WO2015127158A1,US20150203591A1, US20140322221A1, US20130303396A1, US20110293613,US20130017200A1, US20160102135A1, WO2015197598A2, WO2015197582A1, U.S.Pat. No. 9,359,437, US20150018529, WO2016115274A1, WO2016087416A1,US20080069820A1, U.S. Pat. Nos. 9,145,588B, 7,919,257, andUS20150232560A1. Exemplary multispecific molecules utilizing a fullantibody-Fab/scFab format include those described in the following, U.S.Pat. No. 9,382,323B2, US20140072581A1, US20140308285A1, US20130165638A1,US20130267686A1, US20140377269A1, U.S. Pat. No. 7,741,446B2, andWO1995009917A1. Exemplary multispecific molecules utilizing a domainexchange format include those described in the following,US20150315296A1, WO2016087650A1, US20160075785A1, WO2016016299A1,US20160130347A1, US20150166670, U.S. Pat. No. 8,703,132B2,US20100316645, U.S. Pat. No. 8,227,577B2, US20130078249.

Fc-Containing Entities (Mini-Antibodies)

Fc-containing entities, also known as mini-antibodies, can be generatedby fusing scFv to the C-termini of constant heavy region domain 3(CH3-scFv) and/or to the hinge region (scFv-hinge-Fc) of an antibodywith a different specificity. Trivalent entities can also be made whichhave disulfide stabilized variable domains (without peptide linker)fused to the C-terminus of CH3 domains of IgGs.

Fc-Containing Multispecific Molecules

In some embodiments, the multispecific molecules disclosed hereinincludes an immunoglobulin constant region (e.g., an Fc region).Exemplary Fc regions can be chosen from the heavy chain constant regionsof IgG1, IgG2, IgG3 or IgG4; more particularly, the heavy chain constantregion of human IgG1, IgG2, IgG3, or IgG4.

In some embodiments, the immunoglobulin chain constant region (e.g., theFc region) is altered, e.g., mutated, to increase or decrease one ormore of: Fc receptor binding, antibody glycosylation, the number ofcysteine residues, effector cell function, or complement function.

In other embodiments, an interface of a first and second immunoglobulinchain constant regions (e.g., a first and a second Fc region) isaltered, e.g., mutated, to increase or decrease dimerization, e.g.,relative to a non-engineered interface, e.g., a naturally-occurringinterface. For example, dimerization of the immunoglobulin chainconstant region (e.g., the Fc region) can be enhanced by providing an Fcinterface of a first and a second Fc region with one or more of: apaired protuberance-cavity (“knob-in-a hole”), an electrostaticinteraction, or a strand-exchange, such that a greater ratio ofheteromultimer to homomultimer forms, e.g., relative to a non-engineeredinterface.

In some embodiments, the multispecific molecules include a paired aminoacid substitution at a position chosen from one or more of 347, 349,350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409,e.g., of the Fc region of human IgG1 For example, the immunoglobulinchain constant region (e.g., Fc region) can include a paired an aminoacid substitution chosen from: T366S, L368A, or Y407V (e.g.,corresponding to a cavity or hole), and T366W (e.g., corresponding to aprotuberance or knob).

In other embodiments, the multifunctional molecule includes a half-lifeextender, e.g., a human serum albumin or an antibody molecule to humanserum albumin.

Heterodimerized Antibody Molecules & Methods of Making

Various methods of producing multispecific antibodies have beendisclosed to address the problem of incorrect heavy chain pairing.Exemplary methods are described below. Exemplary multispecific antibodyformats and methods of making said multispecific antibodies are alsodisclosed in e.g., Speiss et al. Molecular Immunology 67 (2015) 95-106;and Klein et al mAbs 4:6, 653-663; November/December 2012; the entirecontents of each of which are incorporated by reference herein.

Heterodimerized bispecific antibodies are based on the natural IgGstructure, wherein the two binding arms recognize different antigens.IgG derived formats that enable defined monovalent (and simultaneous)antigen binding are generated by forced heavy chain heterodimerization,combined with technologies that minimize light chain mispairing (e.g.,common light chain). Forced heavy chain heterodimerization can beobtained using, e.g., knob-in-hole OR strand exchange engineered domains(SEED).

Knob-in-Hole

Knob-in-Hole as described in U.S. Pat. Nos. 5,731,116, 7,476,724 andRidgway, J. et al. (1996) Prot. Engineering 9(7): 617-621, broadlyinvolves: (1) mutating the CH3 domain of one or both antibodies topromote heterodimerization; and (2) combining the mutated antibodiesunder conditions that promote heterodimerization. “Knobs” or“protuberances” are typically created by replacing a small amino acid ina parental antibody with a larger amino acid (e.g., T366Y or T366W);“Holes” or “cavities” are created by replacing a larger residue in aparental antibody with a smaller amino acid (e.g., Y407T, T366S, L368Aand/or Y407V).

For bispecific antibodies including an Fc domain, introduction ofspecific mutations into the constant region of the heavy chains topromote the correct heterodimerization of the Fc portion can beutilized. Several such techniques are reviewed in Klein et al. (mAbs(2012) 4:6, 1-11), the contents of which are incorporated herein byreference in their entirety. These techniques include the“knobs-into-holes” (KiH) approach which involves the introduction of abulky residue into one of the CH3 domains of one of the antibody heavychains. This bulky residue fits into a complementary “hole” in the otherCH3 domain of the paired heavy chain so as to promote correct pairing ofheavy chains (see e.g., U.S. Pat. No. 7,642,228).

Exemplary KiH mutations include S354C, T366W in the “knob” heavy chainand Y349C, T366S, L368A, Y407V in the “hole” heavy chain. Otherexemplary KiH mutations are provided in Table 4, with additionaloptional stabilizing Fc cysteine mutations.

TABLE 4 Exemplary Fc KiH mutations and optional Cysteine mutationsPosition Knob Mutation Hole Mutation T366 T366W T366S L368 — L368A Y407— Y407V Additional Cysteine Mutations to form a stabilizing disulfidebridge Position Knob CH3 Hole CH3 S354 S354C — Y349 — Y349C

Other Fc mutations are provided by Igawa and Tsunoda who identified 3negatively charged residues in the CH3 domain of one chain that pairwith three positively charged residues in the CH3 domain of the otherchain. These specific charged residue pairs are: E356-K439, E357-K370,D399-K409 and vice versa. By introducing at least two of the followingthree mutations in chain A: E356K, E357K and D399K, as well as K370E,K409D, K439E in chain B, alone or in combination with newly identifieddisulfide bridges, they were able to favor very efficientheterodimerization while suppressing homodimerization at the same time(Martens T et al. A novel one-armed antic-Met antibody inhibitsglioblastoma growth in vivo. Clin Cancer Res 2006; 12:6144-52;PMID:17062691). Xencor defined 41 variant pairs based on combiningstructural calculations and sequence information that were subsequentlyscreened for maximal heterodimerization, defining the combination ofS364H, F405A (HA) on chain A and Y349T, T394F on chain B (TF) (Moore G Let al. A novel bispecific antibody format enables simultaneous bivalentand monovalent co-engagement of distinct target antigens. MAbs 2011;3:546-57; PMID: 22123055).

Other exemplary Fc mutations to promote heterodimerization ofmultispecific antibodies include those described in the followingreferences, the contents of each of which is incorporated by referenceherein, WO2016071377A1, US20140079689A1, US20160194389A1, US20160257763,WO2016071376A2, WO2015107026A1, WO2015107025A1, WO2015107015A1,US20150353636A1, US20140199294A1, U.S. Pat. No. 7,750,128B2,US20160229915A1, US20150344570A1, U.S. Pat. No. 8,003,774A1,US20150337049A1, US20150175707A1, US20140242075A1, US20130195849A1,US20120149876A1, US20140200331A1, U.S. Pat. No. 9,309,311B2, U.S. Pat.No. 8,586,713, US20140037621A1, US20130178605A1, US20140363426A1,US20140051835A1 and US20110054151A1.

Stabilizing cysteine mutations have also been used in combination withKiH and other Fc heterodimerization promoting variants, see e.g., U.S.Pat. No. 7,183,076. Other exemplary cysteine modifications include,e.g., those disclosed in US20140348839A1, U.S. Pat. No. 7,855,275B2, andU.S. Pat. No. 9,000,130B2.

Strand Exchange Engineered Domains (SEED)

Heterodimeric Fc platform that support the design of bispecific andasymmetric fusion proteins by devising strand-exchange engineered domain(SEED) C(H)3 heterodimers are known. These derivatives of human IgG andIgA C(H)3 domains create complementary human SEED C(H)3 heterodimersthat are composed of alternating segments of human IgA and IgG C(H)3sequences. The resulting pair of SEED C(H)3 domains preferentiallyassociates to form heterodimers when expressed in mammalian cells.SEEDbody (Sb) fusion proteins consist of [IgG1 hinge]-C(H)2-[SEEDC(H)3], that may be genetically linked to one or more fusion partners(see e.g., Davis J H et al. SEEDbodies: fusion proteins based on strandexchange engineered domain (SEED) CH3 heterodimers in an Fc analogueplatform for asymmetric binders or immunofusions and bispecificantibodies. Protein Eng Des Sel 2010; 23:195-202; PMID:20299542 and U.S.Pat. No. 8,871,912. The contents of each of which are incorporated byreference herein).

Duobody

“Duobody” technology to produce bispecific antibodies with correct heavychain pairing are known. The DuoBody technology involves three basicsteps to generate stable bispecific human IgG1 antibodies in apost-production exchange reaction. In a first step, two IgG1s, eachcontaining single matched mutations in the third constant (CH3) domain,are produced separately using standard mammalian recombinant cell lines.Subsequently, these IgG1 antibodies are purified according to standardprocesses for recovery and purification. After production andpurification (post-production), the two antibodies are recombined undertailored laboratory conditions resulting in a bispecific antibodyproduct with a very high yield (typically >95%) (see e.g., Labrijn etal, PNAS 2013; 110(13):5145-5150 and Labrijn et al. Nature Protocols2014; 9(10):2450-63, the contents of each of which are incorporated byreference herein).

Electrostatic Interactions

Methods of making multispecific antibodies using CH3 amino acid changeswith charged amino acids such that homodimer formation iselectrostatically unfavorable are disclosed. EP1870459 and WO 2009089004describe other strategies for favoring heterodimer formation uponco-expression of different antibody domains in a host cell. In thesemethods, one or more residues that make up the heavy chain constantdomain 3 (CH3), CH3-CH3 interfaces in both CH3 domains are replaced witha charged amino acid such that homodimer formation is electrostaticallyunfavorable and heterodimerization is electrostatically favorable.Additional methods of making multispecific molecules using electrostaticinteractions are described in the following references, the contents ofeach of which is incorporated by reference herein, includeUS20100015133, U.S. Pat. No. 8,592,562B2, U.S. Pat. No. 9,200,060B2,US20140154254A1, and U.S. Pat. No. 9,358,286A1.

Common Light Chain

Light chain mispairing needs to be avoided to generate homogenouspreparations of bispecific IgGs. One way to achieve this is through theuse of the common light chain principle, i.e. combining two binders thatshare one light chain but still have separate specificities. Anexemplary method of enhancing the formation of a desired bispecificantibody from a mixture of monomers is by providing a common variablelight chain to interact with each of the heteromeric variable heavychain regions of the bispecific antibody. Compositions and methods ofproducing bispecific antibodies with a common light chain as disclosedin, e.g., U.S. Pat. No. 7,183,076B2, US20110177073A1, EP2847231A1,WO2016079081A1, and EP3055329A1, the contents of each of which isincorporated by reference herein.

CrossMab

Another option to reduce light chain mispairing is the CrossMabtechnology which avoids non-specific L chain mispairing by exchangingCH1 and CL domains in the Fab of one half of the bispecific antibody.Such crossover variants retain binding specificity and affinity, butmake the two arms so different that L chain mispairing is prevented. TheCrossMab technology (as reviewed in Klein et al. Supra) involves domainswapping between heavy and light chains so as to promote the formationof the correct pairings. Briefly, to construct a bispecific IgG-likeCrossMab antibody that could bind to two antigens by using two distinctlight chain-heavy chain pairs, a two-step modification process isapplied. First, a dimerization interface is engineered into theC-terminus of each heavy chain using a heterodimerization approach,e.g., Knob-into-hole (KiH) technology, to ensure that only a heterodimerof two distinct heavy chains from one antibody (e.g., Antibody A) and asecond antibody (e.g., Antibody B) is efficiently formed. Next, theconstant heavy 1 (CH1) and constant light (CL) domains of one antibodyare exchanged (Antibody A), keeping the variable heavy (VH) and variablelight (VL) domains consistent. The exchange of the CH1 and CL domainsensured that the modified antibody (Antibody A) light chain would onlyefficiently dimerize with the modified antibody (antibody A) heavychain, while the unmodified antibody (Antibody B) light chain would onlyefficiently dimerize with the unmodified antibody (Antibody B) heavychain; and thus only the desired bispecific CrossMab would beefficiently formed (see e.g., Cain, C. SciBX 4(28);doi:10.1038/scibx.2011.783, the contents of which are incorporated byreference herein).

Common Heavy Chain

An exemplary method of enhancing the formation of a desired bispecificantibody from a mixture of monomers is by providing a common variableheavy chain to interact with each of the heteromeric variable lightchain regions of the bispecific antibody. Compositions and methods ofproducing bispecific antibodies with a common heavy chain are disclosedin, e.g., US20120184716, US20130317200, and US20160264685A1, thecontents of each of which is incorporated by reference herein.

Amino Acid Modifications

Alternative compositions and methods of producing multispecificantibodies with correct light chain pairing include various amino acidmodifications. For example, Zymeworks describes heterodimers with one ormore amino acid modifications in the CH1 and/or CL domains, one or moreamino acid modifications in the VH and/or VL domains, or a combinationthereof, which are part of the interface between the light chain andheavy chain and create preferential pairing between each heavy chain anda desired light chain such that when the two heavy chains and two lightchains of the heterodimer pair are co-expressed in a cell, the heavychain of the first heterodimer preferentially pairs with one of thelight chains rather than the other (see e.g., WO2015181805). Otherexemplary methods are described in WO2016026943 (Argen-X),US20150211001, US20140072581A1, US20160039947A1, and US20150368352.

Lambda/Kappa Formats

Multispecific molecules (e.g., multispecific antibody molecules) thatinclude the lambda light chain polypeptide and a kappa light chainpolypeptides, can be used to allow for heterodimerization. Methods forgenerating bispecific antibody molecules comprising the lambda lightchain polypeptide and a kappa light chain polypeptides are disclosed inPCT/US17/53053 filed on Sep. 22, 2017 and designated publication numberWO 2018/057955, incorporated herein by reference in its entirety.

In embodiments, the multispecific molecule includes a multispecificantibody molecule, e.g., an antibody molecule comprising two bindingspecificities, e.g., a bispecific antibody molecule. The multispecificantibody molecule includes:

-   -   a lambda light chain polypeptide 1 (LLCP1) specific for a first        epitope;    -   a heavy chain polypeptide 1 (HCP1) specific for the first        epitope;    -   a kappa light chain polypeptide 2 (KLCP2) specific for a second        epitope; and    -   a heavy chain polypeptide 2 (HCP2) specific for the second        epitope.

“Lambda light chain polypeptide 1 (LLCP1)”, as that term is used herein,refers to a polypeptide comprising sufficient light chain (LC) sequence,such that when combined with a cognate heavy chain variable region, canmediate specific binding to its epitope and complex with an HCP1. In anembodiment it comprises all or a fragment of a CH1 region. In anembodiment, an LLCP1 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3,FR4, and CH1, or sufficient sequence therefrom to mediate specificbinding of its epitope and complex with an HCP1. LLCP1, together withits HCP1, provide specificity for a first epitope (while KLCP2, togetherwith its HCP2, provide specificity for a second epitope). As describedelsewhere herein, LLCP1 has a higher affinity for HCP1 than for HCP2.

“Kappa light chain polypeptide 2 (KLCP2)”, as that term is used herein,refers to a polypeptide comprising sufficient light chain (LC) sequence,such that when combined with a cognate heavy chain variable region, canmediate specific binding to its epitope and complex with an HCP2. Insome embodiments, it comprises all or a fragment of a CH1 region. In anembodiment, a KLCP2 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3,FR4, and CH1, or sufficient sequence therefrom to mediate specificbinding of its epitope and complex with an HCP2. KLCP2, together withits HCP2, provide specificity for a second epitope (while LLCP1,together with its HCP1, provide specificity for a first epitope).

“Heavy chain polypeptide 1 (HCP1)”, as that term is used herein, refersto a polypeptide comprising sufficient heavy chain (HC) sequence, e.g.,HC variable region sequence, such that when combined with a cognateLLCP1, can mediate specific binding to its epitope and complex with anHCP1. In some embodiments, it comprises all or a fragment of aCHlregion. In an embodiment, it comprises all or a fragment of a CH2and/or CH3 region. In an embodiment an HCP1 comprises HC-CDR1, HC-CDR2,HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sufficient sequencetherefrom to: (i) mediate specific binding of its epitope and complexwith an LLCP1, (ii) to complex preferentially, as described herein toLLCP1 as opposed to KLCP2; and (iii) to complex preferentially, asdescribed herein, to an HCP2, as opposed to another molecule of HCP1.HCP1, together with its LLCP1, provide specificity for a first epitope(while KLCP2, together with its HCP2, provide specificity for a secondepitope).

“Heavy chain polypeptide 2 (HCP2)”, as that term is used herein, refersto a polypeptide comprising sufficient heavy chain (HC) sequence, e.g.,HC variable region sequence, such that when combined with a cognateLLCP1, can mediate specific binding to its epitope and complex with anHCP1. In some embodiments, it comprises all or a fragment of a CH1region. In some embodiments, it comprises all or a fragment of a CH2and/or CH3 region. In an embodiment an HCP1 comprises HC-CDR1, HC-CDR2,HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sufficient sequencetherefrom to: (i) mediate specific binding of its epitope and complexwith an KLCP2, (ii) to complex preferentially, as described herein toKLCP2 as opposed to LLCP1; and (iii) to complex preferentially, asdescribed herein, to an HCP1, as opposed to another molecule of HCP2.HCP2, together with its KLCP2, provide specificity for a second epitope(while LLCP1, together with its HCP1, provide specificity for a firstepitope).

In some embodiments of the multispecific antibody molecule disclosedherein: LLCP1 has a higher affinity for HCP1 than for HCP2; and/or KLCP2has a higher affinity for HCP2 than for HCP1.

In embodiments, the affinity of LLCP1 for HCP1 is sufficiently greaterthan its affinity for HCP2, such that under preselected conditions,e.g., in aqueous buffer, e.g., at pH 7, in saline, e.g., at pH 7, orunder physiological conditions, at least 75, 80, 90, 95, 98, 99, 99.5,or 99.9% of the multispecific antibody molecule molecules have a LLCP1complexed, or interfaced with, a HCP.

In some embodiments of the multispecific antibody molecule disclosedherein:

-   -   the HCP1 has a greater affinity for HCP2, than for a second        molecule of HCP1; and/or    -   the HCP2 has a greater affinity for HCP1, than for a second        molecule of HCP2.

In embodiments, the affinity of HCP1 for HCP2 is sufficiently greaterthan its affinity for a second molecule of HCP1, such that underpreselected conditions, e.g., in aqueous buffer, e.g., at pH 7, insaline, e.g., at pH 7, or under physiological conditions, at least 75%,80, 90, 95, 98, 99 99.5 or 99.9% of the multispecific antibody moleculemolecules have a HCPlcomplexed, or interfaced with, a HCP2.

In another aspect, disclosed herein is a method for making, orproducing, a multispecific antibody molecule. The method includes:

(i) providing a first heavy chain polypeptide (e.g., a heavy chainpolypeptide comprising one, two, three or all of a first heavy chainvariable region (first VH), a first CH1, a first heavy chain constantregion (e.g., a first CH2, a first CH3, or both));(ii) providing a second heavy chain polypeptide (e.g., a heavy chainpolypeptide comprising one, two, three or all of a second heavy chainvariable region (second VH), a second CH1, a second heavy chain constantregion (e.g., a second CH2, a second CH3, or both));(iii) providing a lambda chain polypeptide (e.g., a lambda lightvariable region (VLλ), a lambda light constant chain (VLλ), or both)that preferentially associates with the first heavy chain polypeptide(e.g., the first VH); and(iv) providing a kappa chain polypeptide (e.g., a lambda light variableregion (VLK), a lambda light constant chain (VLκ), or both) thatpreferentially associates with the second heavy chain polypeptide (e.g.,the second VH), under conditions where (i)-(iv) associate.

In embodiments, the first and second heavy chain polypeptides form an Fcinterface that enhances heterodimerization.

In embodiments, (i)-(iv) (e.g., nucleic acid encoding (i)-(iv)) areintroduced in a single cell, e.g., a single mammalian cell, e.g., a CHOcell. In embodiments, (i)-(iv) are expressed in the cell.

In embodiments, (i)-(iv) (e.g., nucleic acid encoding (i)-(iv)) areintroduced in different cells, e.g., different mammalian cells, e.g.,two or more CHO cell. In embodiments, (i)-(iv) are expressed in thecells.

In embodiments, the method further comprises purifying a cell-expressedantibody molecule, e.g., using a lambda- and/or -kappa-specificpurification, e.g., affinity chromatography.

In embodiments, the method further comprises evaluating thecell-expressed multispecific antibody molecule. For example, thepurified cell-expressed multispecific antibody molecule can be analyzedby techniques known in the art, include mass spectrometry. In oneembodiment, the purified cell-expressed antibody molecule is cleaved,e.g., digested with papain to yield the Fab moieties and evaluated usingmass spectrometry.

In embodiments, the method produces correctly paired kappa/lambdamultispecific, e.g., bispecific, antibody molecules in a high yield,e.g., at least 75%, 80, 90, 95, 98, 99 99.5 or 99.9%.

In other embodiments, the multispecific, e.g., a bispecific, antibodymolecule that includes:

(i) a first heavy chain polypeptide (HCP1) (e.g., a heavy chainpolypeptide comprising one, two, three or all of a first heavy chainvariable region (first VH), a first CH1, a first heavy chain constantregion (e.g., a first CH2, a first CH3, or both)), e.g., wherein theHCP1 binds to a first epitope;(ii) a second heavy chain polypeptide (HCP2) (e.g., a heavy chainpolypeptide comprising one, two, three or all of a second heavy chainvariable region (second VH), a second CH1, a second heavy chain constantregion (e.g., a second CH2, a second CH3, or both)), e.g., wherein theHCP2 binds to a second epitope;(iii) a lambda light chain polypeptide (LLCP1) (e.g., a lambda lightvariable region (VLl), a lambda light constant chain (VLl), or both)that preferentially associates with the first heavy chain polypeptide(e.g., the first VH), e.g., wherein the LLCP1 binds to a first epitope;and(iv) a kappa light chain polypeptide (KLCP2) (e.g., a lambda lightvariable region (VLk), a lambda light constant chain (VLk), or both)that preferentially associates with the second heavy chain polypeptide(e.g., the second VH), e.g., wherein the KLCP2 binds to a secondepitope.

In embodiments, the first and second heavy chain polypeptides form an Fcinterface that enhances heterodimerization. In embodiments, themultispecific antibody molecule has a first binding specificity thatincludes a hybrid VLl-CLl heterodimerized to a first heavy chainvariable region connected to the Fc constant, CH2-CH3 domain (having aknob modification) and a second binding specificity that includes ahybrid VLk-CLk heterodimerized to a second heavy chain variable regionconnected to the Fc constant, CH2-CH3 domain (having a holemodification).

Cytokine Molecules

Cytokines are generally polypeptides that influence cellular activity,for example, through signal transduction pathways. Accordingly, acytokine of the multispecific or multifunctional polypeptide is usefuland can be associated with receptor-mediated signaling that transmits asignal from outside the cell membrane to modulate a response within thecell. Cytokines are proteinaceous signaling compounds that are mediatorsof the immune response. They control many different cellular functionsincluding proliferation, differentiation and cell survival/apoptosis;cytokines are also involved in several pathophysiological processesincluding viral infections and autoimmune diseases. Cytokines aresynthesized under various stimuli by a variety of cells of both theinnate (monocytes, macrophages, dendritic cells) and adaptive (T- andB-cells) immune systems. Cytokines can be classified into two groups:pro- and anti-inflammatory. Pro-inflammatory cytokines, including IFNγ,IL-1, IL-6 and TNF-alpha, are predominantly derived from the innateimmune cells and Th1 cells. Anti-inflammatory cytokines, includingIL-10, IL-4, IL-13 and IL-5, are synthesized from Th2 immune cells.

The present disclosure provides, inter alia, multispecific (e.g., bi-,tri-, quad-specific) or multifunctional molecules, that include, e.g.,are engineered to contain, one or more cytokine molecules, e.g.,immunomodulatory (e.g., proinflammatory) cytokines and variants, e.g.,functional variants, thereof. Accordingly, in some embodiments, thecytokine molecule is an interleukin or a variant, e.g., a functionalvariant thereof. In some embodiments the interleukin is aproinflammatory interleukin. In some embodiments the interleukin ischosen from interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15(IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-7(IL-7), or interferon gamma. In some embodiments, the cytokine moleculeis a proinflammatory cytokine.

In certain embodiments, the cytokine is a single chain cytokine. Incertain embodiments, the cytokine is a multichain cytokine (e.g., thecytokine comprises 2 or more (e.g., 2) polypeptide chains. An exemplarymultichain cytokine is IL-12.

Examples of useful cytokines include, but are not limited to, GM-CSF,IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12,IL-21, IFN-α, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNFβ. In oneembodiment the cytokine of the multispecific or multifunctionalpolypeptide is a cytokine selected from the group of GM-CSF, IL-2, IL-7,IL-8, IL-10, IL-12, IL-15, IL-21, IFN-α, IFN-γ, MIP-1α, MIP-1β andTGF-β. In one embodiment the cytokine of the i the multispecific ormultifunctional polypeptide is a cytokine selected from the group ofIL-2, IL-7, IL-10, IL-12, IL-15, IFN-α, and IFN-γ. In certainembodiments the cytokine is mutated to remove N- and/or O-glycosylationsites. Elimination of glycosylation increases homogeneity of the productobtainable in recombinant production.

In one embodiment, the cytokine of the multispecific or multifunctionalpolypeptide is IL-2. In a specific embodiment, the IL-2 cytokine canelicit one or more of the cellular responses selected from the groupconsisting of: proliferation in an activated T lymphocyte cell,differentiation in an activated T lymphocyte cell, cytotoxic T cell(CTL) activity, proliferation in an activated B cell, differentiation inan activated B cell, proliferation in a natural killer (NK) cell,differentiation in a NK cell, cytokine secretion by an activated T cellor an NK cell, and NK/lymphocyte activated killer (LAK) antitumorcytotoxicity. In another particular embodiment the IL-2 cytokine is amutant IL-2 cytokine having reduced binding affinity to the.alpha.-subunit of the IL-2 receptor. Together with the .beta.- and.gamma.-subunits (also known as CD122 and CD132, respectively), the.alpha.-subunit (also known as CD25) forms the heterotrimerichigh-affinity IL-2 receptor, while the dimeric receptor consisting onlyof the β- and γ-subunits is termed the intermediate-affinity IL-2receptor. As described in PCT patent application numberPCT/EP2012/051991, which is incorporated herein by reference in itsentirety, a mutant IL-2 polypeptide with reduced binding to the.alpha.-subunit of the IL-2 receptor has a reduced ability to induceIL-2 signaling in regulatory T cells, induces less activation-inducedcell death (AICD) in T cells, and has a reduced toxicity profile invivo, compared to a wild-type IL-2 polypeptide. The use of such ancytokine with reduced toxicity is particularly advantageous in amultispecific or multifunctional polypeptide according to the invention,having a long serum half-life due to the presence of an Fc domain. Inone embodiment, the mutant IL-2 cytokine of the multispecific ormultifunctional polypeptide according to the invention comprises atleast one amino acid mutation that reduces or abolishes the affinity ofthe mutant IL-2 cytokine to the .alpha.-subunit of the IL-2 receptor(CD25) but preserves the affinity of the mutant IL-2 cytokine to theintermediate-affinity IL-2 receptor (consisting of the β and γ subunitsof the IL-2 receptor), compared to the non-mutated IL-2 cytokine. In oneembodiment the one or more amino acid mutations are amino acidsubstitutions. In a specific embodiment, the mutant IL-2 cytokinecomprises one, two or three amino acid substitutions at one, two orthree position(s) selected from the positions corresponding to residue42, 45, and 72 of human IL-2. In a more specific embodiment, the mutantIL-2 cytokine comprises three amino acid substitutions at the positionscorresponding to residue 42, 45 and 72 of human IL-2. In an even morespecific embodiment, the mutant IL-2 cytokine is human IL-2 comprisingthe amino acid substitutions F42A, Y45A and L72G. In one embodiment themutant IL-2 cytokine additionally comprises an amino acid mutation at aposition corresponding to position 3 of human IL-2, which eliminates the0-glycosylation site of IL-2. Particularly, said additional amino acidmutation is an amino acid substitution replacing a threonine residue byan alanine residue. A particular mutant IL-2 cytokine useful in theinvention comprises four amino acid substitutions at positionscorresponding to residues 3, 42, 45 and 72 of human IL-2. Specific aminoacid substitutions are T3A, F42A, Y45A and L72G. As demonstrated in PCTpatent application number PCT/EP2012/051991 and in the appendedExamples, said quadruple mutant IL-2 polypeptide (IL-2 qm) exhibits nodetectable binding to CD25, reduced ability to induce apoptosis in Tcells, reduced ability to induce IL-2 signaling in T.sub.reg cells, anda reduced toxicity profile in vivo. However, it retains ability toactivate IL-2 signaling in effector cells, to induce proliferation ofeffector cells, and to generate IFN-γ as a secondary cytokine by NKcells.

The IL-2 or mutant IL-2 cytokine according to any of the aboveembodiments may comprise additional mutations that provide furtheradvantages such as increased expression or stability. For example, thecysteine at position 125 may be replaced with a neutral amino acid suchas alanine, to avoid the formation of disulfide-bridged IL-2 dimers.Thus, in certain embodiments the IL-2 or mutant IL-2 cytokine of themultispecific or multifunctional polypeptide according to the inventioncomprises an additional amino acid mutation at a position correspondingto residue 125 of human IL-2. In one embodiment said additional aminoacid mutation is the amino acid substitution C125A.

In a specific embodiment the IL-2 cytokine of the multispecific ormultifunctional polypeptide comprises the polypeptide sequence of SEQ IDNO: 2270

[APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT].

In another specific embodiment the IL-2 cytokine of the multispecific ormultifunctional polypeptide comprises the polypeptide sequence of SEQ IDNO: 2280

[APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT].

In another embodiment the cytokine of the multispecific ormultifunctional polypeptide is IL-12. In a specific embodiment saidIL-12 cytokine is a single chain IL-12 cytokine. In an even morespecific embodiment the single chain IL-12 cytokine comprises thepolypeptide sequence of SEQ ID NO: 2290

[IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLN AS].

In one embodiment, the IL-12 cytokine can elicit one or more of thecellular responses selected from the group consisting of: proliferationin a NK cell, differentiation in a NK cell, proliferation in a T cell,and differentiation in a T cell.

In another embodiment the cytokine of the multispecific ormultifunctional polypeptide is IL-10. In a specific embodiment saidIL-10 cytokine is a single chain IL-10 cytokine. In an even morespecific embodiment the single chain IL-10 cytokine comprises thepolypeptide sequence of SEQ ID NO: 2300

[SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGGSGGGGSGGGGSGGGGSSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFIN YIEAYMTMKIRN].

In another specific embodiment the IL-10 cytokine is a monomeric IL-10cytokine. In a more specific embodiment the monomeric IL-10 cytokinecomprises the polypeptide sequence of SEQ ID NO: 2310

[SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN].

In one embodiment, the IL-10 cytokine can elicit one or more of thecellular responses selected from the group consisting of: inhibition ofcytokine secretion, inhibition of antigen presentation by antigenpresenting cells, reduction of oxygen radical release, and inhibition ofT cell proliferation. A multispecific or multifunctional polypeptideaccording to the invention wherein the cytokine is IL-10 is particularlyuseful for downregulation of inflammation, e.g. in the treatment of aninflammatory disorder.

In another embodiment, the cytokine of the multispecific ormultifunctional polypeptide is IL-15. In a specific embodiment saidIL-15 cytokine is a mutant IL-15 cytokine having reduced bindingaffinity to the α-subunit of the IL-15 receptor. Without wishing to bebound by theory, a mutant IL-15 polypeptide with reduced binding to the.alpha.-subunit of the IL-15 receptor has a reduced ability to bind tofibroblasts throughout the body, resulting in improved pharmacokineticsand toxicity profile, compared to a wild-type IL-15 polypeptide. The useof an cytokine with reduced toxicity, such as the described mutant IL-2and mutant IL-15 effector moieties, is particularly advantageous in amultispecific or multifunctional polypeptide according to the invention,having a long serum half-life due to the presence of an Fc domain. Inone embodiment the mutant IL-15 cytokine of the multispecific ormultifunctional polypeptide according to the invention comprises atleast one amino acid mutation that reduces or abolishes the affinity ofthe mutant IL-15 cytokine to the .alpha.-subunit of the IL-15 receptorbut preserves the affinity of the mutant IL-15 cytokine to theintermediate-affinity IL-15/IL-2 receptor (consisting of the .beta.- and.gamma.-subunits of the IL-15/IL-2 receptor), compared to thenon-mutated IL-15 cytokine. In one embodiment the amino acid mutation isan amino acid substitution. In a specific embodiment, the mutant IL-15cytokine comprises an amino acid substitution at the positioncorresponding to residue 53 of human IL-15. In a more specificembodiment, the mutant IL-15 cytokine is human IL-15 comprising theamino acid substitution E53A. In one embodiment the mutant IL-15cytokine additionally comprises an amino acid mutation at a positioncorresponding to position 79 of human IL-15, which eliminates theN-glycosylation site of IL-15. Particularly, said additional amino acidmutation is an amino acid substitution replacing an asparagine residueby an alanine residue. In an even more specific embodiment the IL-15cytokine comprises the polypeptide sequence of SEQ ID NO: 2320[NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLASGDASIHDTVENLIILANNSLSSNGAVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS]. In oneembodiment, the IL-15 cytokine can elicit one or more of the cellularresponses selected from the group consisting of: proliferation in anactivated T lymphocyte cell, differentiation in an activated Tlymphocyte cell, cytotoxic T cell (CTL) activity, proliferation in anactivated B cell, differentiation in an activated B cell, proliferationin a natural killer (NK) cell, differentiation in a NK cell, cytokinesecretion by an activated T cell or an NK cell, and NK/lymphocyteactivated killer (LAK) antitumor cytotoxicity.

Mutant cytokine molecules useful as effector moieties in themultispecific or multifunctional polypeptide can be prepared bydeletion, substitution, insertion or modification using genetic orchemical methods well known in the art. Genetic methods may includesite-specific mutagenesis of the encoding DNA sequence, PCR, genesynthesis, and the like. The correct nucleotide changes can be verifiedfor example by sequencing. Substitution or insertion may involve naturalas well as non-natural amino acid residues. Amino acid modificationincludes well known methods of chemical modification such as theaddition or removal of glycosylation sites or carbohydrate attachments,and the like.

In one embodiment, the cytokine, particularly a single-chain cytokine,of the multispecific or multifunctional polypeptide is GM-CSF. In aspecific embodiment, the GM-CSF cytokine can elicit proliferation and/ordifferentiation in a granulocyte, a monocyte or a dendritic cell. In oneembodiment, the cytokine, particularly a single-chain cytokine, of themultispecific or multifunctional polypeptide is IFN-α. In a specificembodiment, the IFN-α cytokine can elicit one or more of the cellularresponses selected from the group consisting of: inhibiting viralreplication in a virus-infected cell, and upregulating the expression ofmajor histocompatibility complex I (MHC I). In another specificembodiment, the IFN-α cytokine can inhibit proliferation in a tumorcell. In one embodiment the cytokine, particularly a single-chaincytokine, of the multispecific or multifunctional polypeptide is IFNγ.In a specific embodiment, the IFN-γ cytokine can elicit one or more ofthe cellular responses selected from the group of: increased macrophageactivity, increased expression of MHC molecules, and increased NK cellactivity. In one embodiment the cytokine, particularly a single-chaincytokine, of the multispecific or multifunctional polypeptide is IL-7.In a specific embodiment, the IL-7 cytokine can elicit proliferation ofT and/or B lymphocytes. In one embodiment, the cytokine, particularly asingle-chain cytokine, of the multispecific or multifunctionalpolypeptide is IL-8. In a specific embodiment, the IL-8 cytokine canelicit chemotaxis in neutrophils. In one embodiment, the cytokine,particularly a single-chain cytokine, of the multispecific ormultifunctional polypeptide, is MIP-1a. In a specific embodiment, theMIP-1a cytokine can elicit chemotaxis in monocytes and T lymphocytecells. In one embodiment, the cytokine, particularly a single-chaincytokine, of the multispecific or multifunctional polypeptide is MIP-1β.In a specific embodiment, the MIP-1β cytokine can elicit chemotaxis inmonocytes and T lymphocyte cells. In one embodiment, the cytokine,particularly a single-chain cytokine, of the multispecific ormultifunctional polypeptide is TGF-3. In a specific embodiment, theTGF-3 cytokine can elicit one or more of the cellular responses selectedfrom the group consisting of: chemotaxis in monocytes, chemotaxis inmacrophages, upregulation of IL-1 expression in activated macrophages,and upregulation of IgA expression in activated B cells.

In one embodiment, the multispecific or multifunctional polypeptide ofthe invention binds to an cytokine receptor with a dissociation constant(K_(D)) that is at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 times greater than that for acontrol cytokine. In another embodiment, the multispecific ormultifunctional polypeptide binds to an cytokine receptor with a K_(D)that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than thatfor a corresponding multispecific or multifunctional polypeptidecomprising two or more effector moieties. In another embodiment, themultispecific or multifunctional polypeptide binds to an cytokinereceptor with a dissociation constant K_(D) that is about 10 timesgreater than that for a corresponding the multispecific ormultifunctional polypeptide comprising two or more cytokines.

In some embodiments, the multispecific molecules disclosed hereininclude a cytokine molecule. In embodiments, the cytokine moleculeincludes a full length, a fragment or a variant of a cytokine; acytokine receptor domain, e.g., a cytokine receptor dimerizing domain;or an agonist of a cytokine receptor, e.g., an antibody molecule (e.g.,an agonistic antibody) to a cytokine receptor.

In some embodiments the cytokine molecule is chosen from IL-2, IL-12,IL-15, IL-18, IL-7, IL-21, or interferon gamma, or a fragment or variantthereof, or a combination of any of the aforesaid cytokines. Thecytokine molecule can be a monomer or a dimer. In embodiments, thecytokine molecule can further include a cytokine receptor dimerizingdomain.

In other embodiments, the cytokine molecule is an agonist of a cytokinereceptor, e.g., an antibody molecule (e.g., an agonistic antibody) to acytokine receptor chosen from an IL-15Ra or IL-21R.

In one embodiment, the cytokine molecule is IL-15, e.g., human IL-15(e.g., comprising the amino acid sequence:

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:2170), a fragment thereof, or an amino acid sequence substantiallyidentical thereto (e.g., 95% to 99.9% identical thereto, or having atleast one amino acid alteration, but not more than five, ten or fifteenalterations (e.g., substitutions, deletions, or insertions, e.g.,conservative substitutions) to the amino acid sequence of SEQ ID NO:2170.

In some embodiments, the cytokine molecule comprises a receptordimerizing domain, e.g., an IL15Ralpha dimerizing domain. In oneembodiment, the IL15Ralpha dimerizing domain comprises the amino acidsequence: MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVL (SEQ ID NO: 2180), a fragment thereof, or an aminoacid sequence substantially identical thereto (e.g., 95% to 99.9%identical thereto, or having at least one amino acid alteration, but notmore than five, ten or fifteen alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) to the aminoacid sequence of SEQ ID NO: 2180. In some embodiments, the cytokinemolecule (e.g., IL-15) and the receptor dimerizing domain (e.g., anIL15Ralpha dimerizing domain) of the multispecific molecule arecovalently linked, e.g., via a linker (e.g., a Gly-Ser linker, e.g., alinker comprising the amino acid sequence SGGSGGGGSGGGSGGGGSLQ (SEQ IDNO: 2190). In other embodiments, the cytokine molecule (e.g., IL-15) andthe receptor dimerizing domain (e.g., an IL15Ralpha dimerizing domain)of the multispecific molecule are not covalently linked, e.g., arenon-covalently associated.

In other embodiments, the cytokine molecule is IL-2, e.g., human IL-2(e.g., comprising the amino acid sequence:APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 2191), a fragment thereof, or an amino acidsequence substantially identical thereto (e.g., 95% to 99.9% identicalthereto, or having at least one amino acid alteration, but not more thanfive, ten or fifteen alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions) to the amino acid sequenceof SEQ ID NO:2191).

In other embodiments, the cytokine molecule is IL-18, e.g., human IL-18(e.g., comprising the amino acid sequence:YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 2192), a fragmentthereof, or an amino acid sequence substantially identical thereto(e.g., 95% to 99.9% identical thereto, or having at least one amino acidalteration, but not more than five, ten or fifteen alterations (e.g.,substitutions, deletions, or insertions, e.g., conservativesubstitutions) to the amino acid sequence of SEQ ID NO: 2192).

In other embodiments, the cytokine molecule is IL-21, e.g., human IL-21(e.g., comprising the amino acid sequence:QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 2193), a fragment thereof, or an amino acidsequence substantially identical thereto (e.g., 95% to 99.9% identicalthereto, or having at least one amino acid alteration, but not more thanfive, ten or fifteen alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions) to the amino acid sequenceof SEQ ID NO: 2193).

In yet other embodiments, the cytokine molecule is interferon gamma,e.g., human interferon gamma (e.g., comprising the amino acid sequence:QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG (SEQ ID NO: 2194), a fragment thereof, or an aminoacid sequence substantially identical thereto (e.g., 95% to 99.9%identical thereto, or having at least one amino acid alteration, but notmore than five, ten or fifteen alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) to the aminoacid sequence of SEQ ID NO: 2194).

Immune Cell Engagers

The immune cell engagers, e.g., first and/or second immune cell engager,of the multispecific or multifunctional molecules disclosed herein canmediate binding to, and/or activation of, an immune cell, e.g., animmune effector cell. In some embodiments, the immune cell is chosenfrom a T cell, an NK cell, a B cell, a dendritic cell, or a macrophagecell engager, or a combination thereof. In some embodiments, the immunecell engager is chosen from one, two, three, or all of a T cell engager,NK cell engager, a B cell engager, a dendritic cell engager, or amacrophage cell engager, or a combination thereof. The immune cellengager can be an agonist of the immune system. In some embodiments, theimmune cell engager can be an antibody molecule, a ligand molecule(e.g., a ligand that further comprises an immunoglobulin constantregion, e.g., an Fc region), a small molecule, a nucleotide molecule.

Natural Killer Cell Engagers

Natural Killer (NK) cells recognize and destroy tumors andvirus-infected cells in an antibody-independent manner. The regulationof NK cells is mediated by activating and inhibiting receptors on the NKcell surface. One family of activating receptors is the naturalcytotoxicity receptors (NCRs) which include NKp30, NKp44 and NKp46. TheNCRs initiate tumor targeting by recognition of heparan sulfate oncancer cells. NKG2D is a receptor that provides both stimulatory andcostimulatory innate immune responses on activated killer (NK) cells,leading to cytotoxic activity. DNAM1 is a receptor involved inintercellular adhesion, lymphocyte signaling, cytotoxicity andlymphokine secretion mediated by cytotoxic T-lymphocyte (CTL) and NKcell. DAP10 (also known as HCST) is a transmembrane adapter proteinwhich associates with KLRK1 to form an activation receptor KLRK1-HCST inlymphoid and myeloid cells; this receptor plays a major role intriggering cytotoxicity against target cells expressing cell surfaceligands such as WIC class I chain-related MICA and MICB, and U(optionally L1)6-binding proteins (ULBPs); it KLRK1-HCST receptor playsa role in immune surveillance against tumors and is required forcytolysis of tumors cells; indeed, melanoma cells that do not expressKLRK1 ligands escape from immune surveillance mediated by NK cells. CD16is a receptor for the Fc region of IgG, which binds complexed oraggregated IgG and also monomeric IgG and thereby mediatesantibody-dependent cellular cytotoxicity (ADCC) and otherantibody-dependent responses, such as phagocytosis.

In some embodiments, the NK cell engager is a viral hemagglutinin (HA),HA is a glycoprotein found on the surface of influenza viruses. It isresponsible for binding the virus to cells with sialic acid on themembranes, such as cells in the upper respiratory tract or erythrocytes.HA has at least 18 different antigens. These subtypes are named H1through H18. NCRs can recognize viral proteins. NKp46 has been shown tobe able to interact with the HA of influenza and the HA-NA ofParamyxovirus, including Sendai virus and Newcastle disease virus.Besides NKp46, NKp44 can also functionally interact with HA of differentinfluenza subtypes.

The present disclosure provides, inter alia, multispecific (e.g., bi-,tri-, quad-specific) or multifunctional molecules, that are engineeredto contain one or more NK cell engagers that mediate binding to and/oractivation of an NK cell. Accordingly, in some embodiments, the NK cellengager is selected from an antigen binding domain or ligand that bindsto (e.g., activates): NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10,CD16 (e.g., CD16a, CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD100(SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7,KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C,NKG2E, or CD160.

In one embodiment, the NK cell engager is a ligand of NKp30 is a B7-6,e.g., comprises the amino acid sequence of:DLKVEMMAGGTQITPLNDNVTIFCNIFYSQPLNITSMGITWFWKSLTFDKEVKVFEFFGDHQEAFRPGAIVSPWRLKSGDASLRLPGIQLEEAGEYRCEVVVTPLKAQGTVQLEVVASPASRLLLDQVGMKENEDKYMCESSGFYPEAINITWEKQTQKFPHPIEISEDVITGPTIKNMDGTFNVTSCLKLNSSQEDPGTVYQCVVRHASLHTPLRSNFTLTAARHSLSETEKTDNFS (SEQ ID NO:3291), a fragment thereof, or an amino acid sequence substantiallyidentical thereto (e.g., 95% to 99.9% identical thereto, or having atleast one amino acid alteration, but not more than five, ten or fifteenalterations (e.g., substitutions, deletions, or insertions, e.g.,conservative substitutions) to the amino acid sequence of SEQ ID NO:3291.

In other embodiments, the NK cell engager is a ligand of NKp44 or NKp46,which is a viral HA. Viral hemagglutinins (HA) are glyco proteins whichare on the surface of viruses. HA proteins allow viruses to bind to themembrane of cells via sialic acid sugar moieties which contributes tothe fusion of viral membranes with the cell membranes (see e.g., Eur JImmunol. 2001 September; 31(9):2680-9 “Recognition of viralhemagglutinins by NKp44 but not by NKp30”; and Nature. 2001 Feb. 22;409(6823):1055-60 “Recognition of haemagglutinins on virus-infectedcells by NKp46 activates lysis by human NK cells” the contents of eachof which are incorporated by reference herein).

In other embodiments, the NK cell engager is a ligand of NKG2D chosenfrom MICA, MICB, or ULBP1, e.g., wherein:

(i) MICA comprises the amino acid sequence:EPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETKEWTMPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHW (SEQ ID NO: 3292),a fragment thereof, or an amino acid sequence substantially identicalthereto (e.g., 95% to 99.9% identical thereto, or having at least oneamino acid alteration, but not more than five, ten or fifteenalterations (e.g., substitutions, deletions, or insertions, e.g.,conservative substitutions) to the amino acid sequence of SEQ ID NO:3292;(ii) MICB comprises the amino acid sequence:AEPHSLRYNLMVLSQDESVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAEDVLGAKTWDTETEDLTENGQDLRRTLTHIKDQKGGLHSLQEIRVCEIHEDSSTRGSRHFYYDGELFLSQNLETQESTVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRAS SFYPRNITLTWRQDGVSLSHNTQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKVLVLQSQRTD (SEQ ID NO:3293), a fragment thereof, or an amino acid sequence substantiallyidentical thereto (e.g., 95% to 99.9% identical thereto, or having atleast one amino acid alteration, but not more than five, ten or fifteenalterations (e.g., substitutions, deletions, or insertions, e.g.,conservative substitutions) to the amino acid sequence of SEQ ID NO:3293; or(iii) ULBP1 comprises the amino acid sequence:GWVDTHCLCYDFIITPKSRPEPQWCEVQGLVDERPFLHYDCVNHKAKAFASLGKKVNVTKTWEEQTETLRDVVDFLKGQLLDIQVENLIPIEPLTLQARMSCEHEAHGHGRGSWQFLFNGQKFLLFDSNNRKWTALHPGAKKMTEKWEKNRDVTMFFQKISLGDCKMWLEEFLMYWEQMLDPTKPPSLAPG (SEQ ID NO: 3294), a fragment thereof, or an aminoacid sequence substantially identical thereto (e.g., 95% to 99.9%identical thereto, or having at least one amino acid alteration, but notmore than five, ten or fifteen alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) to the aminoacid sequence of SEQ ID NO: 3294.

In other embodiments, the NK cell engager is a ligand of DNAM1 chosenfrom NECTIN2 or NECL5, e.g., wherein:

(i) NECTIN2 comprises the amino acid sequence:QDVRVQVLPEVRGQLGGTVELPCHLLPPVPGLYISLVTWQRPDAPANHQNVAAFHPKMGPSFPSPKPGSERLSFVSAKQSTGQDTEAELQDATLALHGLTVEDEGNYTCEFATFPKGSVRGMTWLRVIAKPKNQAEAQKVTFSQDPTTVALCISKEGRPPARISWLSSLDWEAKETQVSGTLAGTVTVTSRFTLVPSGRADGVTVTCKVEHESFEEPALIPVTLSVRYPPEVSISGYDDNWYLGRTDATLSCDVRSNPEPTGYDWSTTSGTFPTSAVAQGSQLVIHAVDSLFNTTFVCTVTNAVGMGRAEQVIFVRETPNTAGAGATGG (SEQ ID NO: 3295), a fragment thereof,or an amino acid sequence substantially identical thereto (e.g., 95% to99.9% identical thereto, or having at least one amino acid alteration,but not more than five, ten or fifteen alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) to the aminoacid sequence of SEQ ID NO: 3295; or(ii) NECL5 comprises the amino acid sequence:WPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGISRN (SEQ ID NO: 3296), a fragment thereof, oran amino acid sequence substantially identical thereto (e.g., 95% to99.9% identical thereto, or having at least one amino acid alteration,but not more than five, ten or fifteen alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) to the aminoacid sequence of SEQ ID NO: 3296.

In yet other embodiments, the NK cell engager is a ligand of DAP10,which is an adapter for NKG2D (see e.g., Proc Natl Acad Sci USA. 2005May 24; 102(21): 7641-7646; and Blood, 15 Sep. 2011 Volume 118, Number11, the full contents of each of which is incorporated by referenceherein).

In other embodiments, the NK cell engager is a ligand of CD16, which isa CD16a/b ligand, e.g., a CD16a/b ligand further comprising an antibodyFc region (see e.g., Front Immunol. 2013; 4: 76 discusses how antibodiesuse the Fc to trigger NK cells through CD16,the full contents of whichare incorporated herein).

In other embodiments, the NK cell engager is a ligand of CRTAM, which isNECL2, e.g., wherein NECL2 comprises the amino acid sequence:QNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCTAMASKPATTIRWFKGNTELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVICQVEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDALELTCEAIGKPQPVMVTWVRVDDEMPQHAVLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDYMLYVYDPPTTIPPPTTTTTTTTTTTTTILTIITDSRAGEEGSIRAVDH (SEQ ID NO: 3297), a fragmentthereof, or an amino acid sequence substantially identical thereto(e.g., 95% to 99.9% identical thereto, or having at least one amino acidalteration, but not more than five, ten or fifteen alterations (e.g.,substitutions, deletions, or insertions, e.g., conservativesubstitutions) to the amino acid sequence of SEQ ID NO: 3297.

In other embodiments, the NK cell engager is a ligand of CD27, which isCD70, e.g., wherein CD70 comprises the amino acid sequence:QRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP (SEQ ID NO: 3298), a fragmentthereof, or an amino acid sequence substantially identical thereto(e.g., 95% to 99.9% identical thereto, or having at least one amino acidalteration, but not more than five, ten or fifteen alterations (e.g.,substitutions, deletions, or insertions, e.g., conservativesubstitutions) to the amino acid sequence of SEQ ID NO: 3298.

In other embodiments, the NK cell engager is a ligand of PSGL1, which isL-selectin (CD62L), e.g., wherein L-selectin comprises the amino acidsequence: WTYHYSEKPMNWQRARRFCRDNYTDLVAIQNKAEIEYLEKTLPFSRSYYWIGIRKIGGIWTWVGTNKSLTEEAENWGDGEPNNKKNKEDCVEIYIKRNKDAGKWNDDACHKLKAALCYTASCQPWSCSGHGECVEIINNYTCNCDVGYYGPQCQFVIQCEPLEAPELGTMDCTHPLGNFSFSSQCAFSCSEGTNLTGIEETTCGPFGNWSSPEPTCQVIQCEPLSAPDLGIMNCSHPLASFSFTSACTFICSEGTELIGKKKTICESSGIWSNPSPICQKLDKSFSMIKEGDYN (SEQ ID NO:3299), a fragment thereof, or an amino acid sequence substantiallyidentical thereto (e.g., 95% to 99.9% identical thereto, or having atleast one amino acid alteration, but not more than five, ten or fifteenalterations (e.g., substitutions, deletions, or insertions, e.g.,conservative substitutions) to the amino acid sequence of SEQ ID NO:3299.

In other embodiments, the NK cell engager is a ligand of CD96, which isNECL5, e.g., wherein NECL5 comprises the amino acid sequence:WPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGISRN (SEQ ID NO: 3296), a fragment thereof, oran amino acid sequence substantially identical thereto (e.g., 95% to99.9% identical thereto, or having at least one amino acid alteration,but not more than five, ten or fifteen alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) to the aminoacid sequence of SEQ ID NO: 3296.

In other embodiments, the NK cell engager is a ligand of CD100 (SEMA4D),which is CD72, e.g., wherein CD72 comprises the amino acid sequence:RYLQVSQQLQQTNRVLEVTNSSLRQQLRLKITQLGQSAEDLQGSRRELAQSQEALQVEQRAHQAAEGQLQACQADRQKTKETLQSEEQQRRALEQKLSNMENRLKPFFTCGSADTCCPSGWIMHQKSCFYISLTSKNWQESQKQCETLSSKLATFSEIYPQSHSYYFLNSLLPNGGSGNSYWTGLSSNKDWKLTDDTQRTRTYAQSSKCNKVHKTWSWWTLESESCRSSLPYICE MTAFRFPD (SEQID NO: 3300), a fragment thereof, or an amino acid sequencesubstantially identical thereto (e.g., 95% to 99.9% identical thereto,or having at least one amino acid alteration, but not more than five,ten or fifteen alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions) to the amino acid sequenceof SEQ ID NO: 3300.

In other embodiments, the NK cell engager is a ligand of NKp80, which isCLEC2B (AICL), e.g., wherein CLEC2B (AICL) comprises the amino acidsequence: KLTRDSQSLCPYDWIGFQNKCYYF SKEEGDWNSSKYNCSTQHADLTIIDNIEEMNFLRRYKCSSDHWIGLKMAKNRTGQWVDGATFTKSFGMRGSEGCAYLSDDGAATARCYTER KWICRKRIH (SEQID NO: 3301), a fragment thereof, or an amino acid sequencesubstantially identical thereto (e.g., 95% to 99.9% identical thereto,or having at least one amino acid alteration, but not more than five,ten or fifteen alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions) to the amino acid sequenceof SEQ ID NO: 3301.

In other embodiments, the NK cell engager is a ligand of CD244, which isCD48, e.g., wherein CD48 comprises the amino acid sequence:QGHLVHMTVVSGSNVTLNISESLPENYKQLTWFYTFDQKIVEWDSRKSKYFESKFKGRVRLDPQSGALYISKVQKEDNSTYIMRVLKKTGNEQEWKIKLQVLDPVPKPVIKIEKIEDMDDNCYLKLSCVIPGESVNYTWYGDKRPFPKELQNSVLETTLMPHNYSRCYTCQVSNSVSSKNGTVCLSPPCTLARS (SEQ ID NO: 3302), a fragment thereof, or an aminoacid sequence substantially identical thereto (e.g., 95% to 99.9%identical thereto, or having at least one amino acid alteration, but notmore than five, ten or fifteen alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) to the aminoacid sequence of SEQ ID NO: 3302.

T Cell Engagers

The present disclosure provides, inter alia, multispecific (e.g., bi-,tri-, quad-specific) or multifunctional molecules, that are engineeredto contain one or more T cell engager that mediate binding to and/oractivation of a T cell. In some embodiments, the T cell engager is anantigen binding domain that binds to, e.g., activates TCR3, e.g., aTCRβV region, as described herein. In some embodiments, the T cellengager is selected from an antigen binding domain or ligand that bindsto (e.g., and in some embodiments activates) one or more of CD3, TCRα,TCRγ, TCR ζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3,GITR, CD30, TIM1, SLAM, CD2, or CD226. In other embodiments, the T cellengager is selected from an antigen binding domain or ligand that bindsto and does not activate one or more of CD3, TCRα, TCRγ, TCR ζ, ICOS,CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM,CD2, or CD226.

B Cell, Macrophage & Dendritic Cell Engagers

Broadly, B cells, also known as B lymphocytes, are a type of white bloodcell of the lymphocyte subtype. They function in the humoral immunitycomponent of the adaptive immune system by secreting antibodies.Additionally, B cells present antigen (they are also classified asprofessional antigen-presenting cells (APCs)) and secrete cytokines.Macrophages are a type of white blood cell that engulfs and digestscellular debris, foreign substances, microbes, cancer cells viaphagocytosis. Besides phagocytosis, they play important roles innonspecific defense (innate immunity) and also help initiate specificdefense mechanisms (adaptive immunity) by recruiting other immune cellssuch as lymphocytes. For example, they are important as antigenpresenters to T cells. Beyond increasing inflammation and stimulatingthe immune system, macrophages also play an important anti-inflammatoryrole and can decrease immune reactions through the release of cytokines.Dendritic cells (DCs) are antigen-presenting cells that function inprocessing antigen material and present it on the cell surface to the Tcells of the immune system.

The present disclosure provides, inter alia, multispecific (e.g., bi-,tri-, quad-specific) or multifunctional molecules, that include, e.g.,are engineered to contain, one or more B cell, macrophage, and/ordendritic cell engager that mediate binding to and/or activation of a Bcell, macrophage, and/or dendritic cell.

Accordingly, in some embodiments, the immune cell engager comprises a Bcell, macrophage, and/or dendritic cell engager chosen from one or moreof CD40 ligand (CD40L) or a CD70 ligand; an antibody molecule that bindsto CD40 or CD70; an antibody molecule to OX40; an OX40 ligand (OX40L);an agonist of a Toll-like receptor (e.g., as described herein, e.g., aTLR4, e.g., a constitutively active TLR4 (caTLR4), or a TLR9 agonists);a 41BB; a CD2; a CD47; or a STING agonist, or a combination thereof.

In some embodiments, the B cell engager is a CD40L, an OX40L, or a CD70ligand, or an antibody molecule that binds to OX40, CD40 or CD70.

In some embodiments, the macrophage engager is a CD2 agonist. In someembodiments, the macrophage engager is an antigen binding domain thatbinds to: CD40L or antigen binding domain or ligand that binds CD40, aToll like receptor (TLR) agonist (e.g., as described herein), e.g., aTLR9 or TLR4 (e.g., caTLR4 (constitutively active TLR4), CD47, or aSTING agonist. In some embodiments, the STING agonist is a cyclicdinucleotide, e.g., cyclic di-GMP (cdGMP) or cyclic di-AMP (cdAMP). Insome embodiments, the STING agonist is biotinylated.

In some embodiments, the dendritic cell engager is a CD2 agonist. Insome embodiments, the dendritic cell engager is a ligand, a receptoragonist, or an antibody molecule that binds to one or more of: OX40L,41BB, a TLR agonist (e.g., as described herein) (e.g., TLR9 agonist,TLR4 (e.g., caTLR4 (constitutively active TLR4)), CD47, or and a STINGagonist. In some embodiments, the STING agonist is a cyclicdinucleotide, e.g., cyclic di-GMP (cdGMP) or cyclic di-AMP (cdAMP). Insome embodiments, the STING agonist is biotinylated.

In other embodiments, the immune cell engager mediates binding to, oractivation of, one or more of a B cell, a macrophage, and/or a dendriticcell. Exemplary B cell, macrophage, and/or dendritic cell engagers canbe chosen from one or more of CD40 ligand (CD40L) or a CD70 ligand; anantibody molecule that binds to CD40 or CD70; an antibody molecule toOX40; an OX40 ligand (OX40L); a Toll-like receptor agonist (e.g., aTLR4, e.g., a constitutively active TLR4 (caTLR4) or a TLR9 agonist); a41BB agonist; a CD2; a CD47; or a STING agonist, or a combinationthereof.

In some embodiments, the B cell engager is chosen from one or more of aCD40L, an OX40L, or a CD70 ligand, or an antibody molecule that binds toOX40, CD40 or CD70.

In other embodiments, the macrophage cell engager is chosen from one ormore of a CD2 agonist; a CD40L; an OX40L; an antibody molecule thatbinds to OX40, CD40 or CD70; a Toll-like receptor agonist or a fragmentthereof (e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4)); aCD47 agonist; or a STING agonist.

In other embodiments, the dendritic cell engager is chosen from one ormore of a CD2 agonist, an OX40 antibody, an OX40L, 41BB agonist, aToll-like receptor agonist or a fragment thereof (e.g., a TLR4, e.g., aconstitutively active TLR4 (caTLR4)), CD47 agonist, or a STING agonist.

In one embodiment, the OX40L comprises the amino acid sequence:QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL (SEQ ID NO: 3303), a fragment thereof, or an amino acidsequence substantially identical thereto (e.g., 95% to 99.9% identicalthereto, or having at least one amino acid alteration, but not more thanfive, ten or fifteen alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions) to the amino acid sequenceof SEQ ID NO: 3303.

In another embodiment, the CD40L comprises the amino acid sequence:MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL (SEQ ID NO: 3304), a fragment thereof,or an amino acid sequence substantially identical thereto (e.g., 95% to99.9% identical thereto, or having at least one amino acid alteration,but not more than five, ten or fifteen alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) to the aminoacid sequence of SEQ ID NO: 3304.

In yet other embodiments, the STING agonist comprises a cyclicdinucleotide, e.g., a cyclic di-GMP (cdGMP), a cyclic di-AMP (cdAMP), ora combination thereof, optionally with 2′,5′ or 3′,5′ phosphatelinkages.

In one embodiment, the immune cell engager includes 41BB ligand, e.g.,comprising the amino acid sequence:ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 3305), a fragment thereof, oran amino acid sequence substantially identical thereto (e.g., 95% to99.9% identical thereto, or having at least one amino acid alteration,but not more than five, ten or fifteen alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) to the aminoacid sequence of SEQ ID NO: 3305.

Toll-Like Receptors

Toll-Like Receptors (TLRs) are evolutionarily conserved receptors arehomologues of the Drosophila Toll protein, and recognize highlyconserved structural motifs known as pathogen-associated microbialpatterns (PAMPs), which are exclusively expressed by microbialpathogens, or danger-associated molecular patterns (DAMPs) that areendogenous molecules released from necrotic or dying cells. PAMPsinclude various bacterial cell wall components such aslipopolysaccharide (LPS), peptidoglycan (PGN) and lipopeptides, as wellas flagellin, bacterial DNA and viral double-stranded RNA. DAMPs includeintracellular proteins such as heat shock proteins as well as proteinfragments from the extracellular matrix. Stimulation of TLRs by thecorresponding PAMPs or DAMPs initiates signaling cascades leading to theactivation of transcription factors, such as AP-1, NF-κB and interferonregulatory factors (IRFs). Signaling by TLRs results in a variety ofcellular responses, including the production of interferons (IFNs),pro-inflammatory cytokines and effector cytokines that direct theadaptive immune response. TLRs are implicated in a number ofinflammatory and immune disorders and play a role in cancer(Rakoff-Nahoum S. & Medzhitov R., 2009. Toll-like receptors and cancer.Nat Revs Cancer 9:57-63.)

TLRs are type I transmembrane proteins characterized by an extracellulardomain containing leucine-rich repeats (LRRs) and a cytoplasmic tailthat contains a conserved region called the Toll/IL-1 receptor (TIR)domain. Ten human and twelve murine TLRs have been characterized, TLR1to TLR10 in humans, and TLR1 to TLR9, TLR11, TLR12 and TLR13 in mice,the homolog of TLR10 being a pseudogene. TLR2 is essential for therecognition of a variety of PAMPs from Gram-positive bacteria, includingbacterial lipoproteins, lipomannans and lipoteichoic acids. TLR3 isimplicated in virus-derived double-stranded RNA. TLR4 is predominantlyactivated by lipopolysaccharide. TLRS detects bacterial flagellin andTLR9 is required for response to unmethylated CpG DNA. Finally, TLR7 andTLR8 recognize small synthetic antiviral molecules, and single-strandedRNA was reported to be their natural ligand. TLR11 has been reported torecognize uropathogenic E. coli and a profilin-like protein fromToxoplasma gondii. The repertoire of specificities of the TLRs isapparently extended by the ability of TLRs to heterodimerize with oneanother. For example, dimers of TLR2 and TLR6 are required for responsesto diacylated lipoproteins while TLR2 and TLR1 interact to recognizetriacylated lipoproteins. Specificities of the TLRs are also influencedby various adapter and accessory molecules, such as MD-2 and CD14 thatform a complex with TLR4 in response to LPS.

TLR signaling consists of at least two distinct pathways: aMyD88-dependent pathway that leads to the production of inflammatorycytokines, and a MyD88-independent pathway associated with thestimulation of IFN-β and the maturation of dendritic cells. TheMyD88-dependent pathway is common to all TLRs, except TLR3 (Adachi O. etal., 1998. Targeted disruption of the MyD88 gene results in loss ofIL-1- and IL-18-mediated function. Immunity. 9(1):143-50). Uponactivation by PAMPs or DAMPs, TLRs hetero- or homodimerize inducing therecruitment of adaptor proteins via the cytoplasmic TIR domain.Individual TLRs induce different signaling responses by usage of thedifferent adaptor molecules. TLR4 and TLR2 signaling requires theadaptor TIRAP/Mal, which is involved in the MyD88-dependent pathway.TLR3 triggers the production of IFN-β in response to double-strandedRNA, in a MyD88-independent manner, through the adaptor TRIF/TICAM-1.TRAM/TICAM-2 is another adaptor molecule involved in theMyD88-independent pathway which function is restricted to the TLR4pathway.

TLR3, TLR7, TLR8 and TLR9 recognize viral nucleic acids and induce typeI IFNs. The signaling mechanisms leading to the induction of type I IFNsdiffer depending on the TLR activated. They involve the interferonregulatory factors, IRFs, a family of transcription factors known toplay a critical role in antiviral defense, cell growth and immuneregulation. Three IRFs (IRF3, IRF5 and IRF7) function as directtransducers of virus-mediated TLR signaling. TLR3 and TLR4 activate IRF3and IRF7, while TLR7 and TLR8 activate IRF5 and IRF7 (Doyle S. et al.,2002. IRF3 mediates a TLR3/TLR4-specific antiviral gene program.Immunity. 17(3):251-63). Furthermore, type I IFN production stimulatedby TLR9 ligand CpG-A has been shown to be mediated by PI(3)K and mTOR(Costa-Mattioli M. & Sonenberg N. 2008. RAPping production of type Iinterferon in pDCs through mTOR. Nature Immunol. 9: 1097-1099).

TLR-9

TLR9 recognizes unmethylated CpG sequences in DNA molecules. CpG sitesare relatively rare (˜1%) on vertebrate genomes in comparison tobacterial genomes or viral DNA. TLR9 is expressed by numerous cells ofthe immune system such as B lymphocytes, monocytes, natural killer (NK)cells, and plasmacytoid dendritic cells. TLR9 is expressedintracellularly, within the endosomal compartments and functions toalert the immune system of viral and bacterial infections by binding toDNA rich in CpG motifs. TLR9 signals leads to activation of the cellsinitiating pro-inflammatory reactions that result in the production ofcytokines such as type-I interferon and IL-12.

TLR Agonists

A TLR agonist can agonize one or more TLR, e.g., one or more of humanTLR-1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, an adjunctiveagent described herein is a TLR agonist. In some embodiments, the TLRagonist specifically agonizes human TLR-9. In some embodiments, theTLR-9 agonist is a CpG moiety. As used herein, a CpG moiety, is a lineardinucleotide having the sequence: 5′-C-phosphate-G-3′, that is, cytosineand guanine separated by only one phosphate.

In some embodiments, the CpG moiety comprises at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, or more CpG dinucleotides. In some embodiments, theCpG moiety consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 CpGdinucleotides. In some embodiments, the CpG moiety has 1-5, 1-10, 1-20,1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 10-20, 10-30, 10-40, or 10-50 CpGdinucleotides.

In some embodiments, the TLR-9 agonist is a synthetic ODN(oligodeoxynucleotides). CpG ODNs are short synthetic single-strandedDNA molecules containing unmethylated CpG dinucleotides in particularsequence contexts (CpG motifs). CpG ODNs possess a partially orcompletely phosphorothioated (PS) backbone, as opposed to the naturalphosphodiester (PO) backbone found in genomic bacterial DNA. There arethree major classes of CpG ODNs: classes A, B and C, which differ intheir immunostimulatory activities. CpG-A ODNs are characterized by a POcentral CpG-containing palindromic motif and a PS-modified 3′ poly-Gstring. They induce high IFN-α production from pDCs but are weakstimulators of TLR9-dependent NF-κB signaling and pro-inflammatorycytokine (e.g. IL-6) production. CpG-B ODNs contain a full PS backbonewith one or more CpG dinucleotides. They strongly activate B cells andTLR9-dependent NF-κB signaling but weakly stimulate IFN-α secretion.CpG-C ODNs combine features of both classes A and B. They contain acomplete PS backbone and a CpG-containing palindromic motif. C-Class CpGODNs induce strong IFN-α production from pDC as well as B cellstimulation.

Tumor-Targeting Moieties

The present disclosure provides, inter alia, multispecific (e.g., bi-,tri-, tetra-specific) molecules, that include, e.g., are engineered tocontain, one or more tumor specific targeting moieties that direct themolecule to a tumor cell.

In certain embodiments, the multispecific molecules disclosed hereininclude a tumor-targeting moiety. The tumor targeting moiety can bechosen from an antibody molecule (e.g., an antigen binding domain asdescribed herein), a receptor or a receptor fragment, or a ligand or aligand fragment, or a combination thereof. In some embodiments, thetumor targeting moiety associates with, e.g., binds to, a tumor cell(e.g., a molecule, e.g., antigen, present on the surface of the tumorcell). In certain embodiments, the tumor targeting moiety targets, e.g.,directs the multispecific molecules disclosed herein to a cancer (e.g.,a cancer or tumor cells). In some embodiments, the cancer is chosen froma hematological cancer, a solid cancer, a metastatic cancer, or acombination thereof.

In some embodiments, the multispecific molecule, e.g., thetumor-targeting moiety, binds to a solid tumor antigen or a stromalantigen. The solid tumor antigen or stromal antigen can be present on asolid tumor, or a metastatic lesion thereof. In some embodiments, thesolid tumor is chosen from one or more of pancreatic (e.g., pancreaticadenocarcinoma), breast, colorectal, lung (e.g., small or non-small celllung cancer), skin, ovarian, or liver cancer. In one embodiment, thesolid tumor is a fibrotic or desmoplastic solid tumor. For example, thesolid tumor antigen or stromal antigen can be present on a tumor, e.g.,a tumor of a class typified by having one or more of: limited tumorperfusion, compressed blood vessels, or fibrotic tumor interstitium.

In certain embodiments, the solid tumor antigen is chosen from one ormore of: PDL1, CD47, gangloside 2 (GD2), prostate stem cell antigen(PSCA), prostate specific membrane antigen (PMSA), prostate-specificantigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met,Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloridechannel 2, Cyclin-B1, 9D7, Ep-CAM, EphA3, Her2/neu, Telomerase, SAP-1,Survivin, NY-ESO-1/LAGE-1, PRAME, SSX-2, Melan-A/MART-1, Gp100/pme117,Tyrosinase, TRP-1/-2, MC1R, β-catenin, BRCA1/2, CDK4, CML66,Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA-125,BAGE, GAGE, NY-ESO-1, 13-catenin, CDK4, CDC27, CD47, a actinin-4,TRP1/gp75, TRP2, gp100, Melan-A/MART1, gangliosides, WT1, EphA3,Epidermal growth factor receptor (EGFR), MART-2, MART-1, MUC1, MUC2,MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, RCC, RUI1, RUI2, SAGE, TRG,TRP1, TSTA, Folate receptor alpha, L1-CAM, CAIX, EGFRvIII, gpA33, GD3,GM2, VEGFR, Intergrins (Integrin alphaVbeta3, Integrin alpha5Beta1),Carbohydrates (Le), IGF1R, EPHA3, TRAILR1, TRAILR2, or RANKL.

In other embodiments, the multispecific molecule, e.g., thetumor-targeting moiety, binds to a molecule, e.g., antigen, present onthe surface of a hematological cancer, e.g., a leukemia or a lymphoma.In some embodiments, the hematological cancer is a B-cell or T cellmalignancy. In some embodiments, the hematological cancer is chosen fromone or more of a Hodgkin's lymphoma, Non-Hodgkin's lymphoma (e.g., Bcell lymphoma, diffuse large B cell lymphoma, follicular lymphoma,chronic lymphocytic leukemia, mantle cell lymphoma, marginal zone B-celllymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cellleukemia), acute myeloid leukemia (AML), chronic myeloid leukemia,myelodysplastic syndrome (MDS), multiple myeloma, or acute lymphocyticleukemia. In embodiments, the cancer is other than acute myeloidleukemia (AML) or myelodysplastic syndrome (MDS). In embodiments, thehematological antigen is chosen from CD47, CD99, CD30, CD38, SLAMF7, orNY-ESO1. In some embodiments, the hematological antigen is chosen fromis chosen from one or more of: BCMA, CD19, CD20, CD22, CD33, CD123,FcRH5, CLEC12, or CD179A.

Stromal Modifying Moieties

Solid tumors have a distinct structure that mimics that of normaltissues and comprises two distinct but interdependent compartments: theparenchyma (neoplastic cells) and the stroma that the neoplastic cellsinduce and in which they are dispersed. All tumors have stroma andrequire stroma for nutritional support and for the removal of wasteproducts. In the case of tumors which grow as cell suspensions (e.g.,leukemias, ascites tumors), the blood plasma serves as stroma (ConnollyJ L et al. Tumor Structure and Tumor Stroma Generation. In: Kufe D W etal., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton: BCDecker; 2003). The stroma includes a variety of cell types, includingfibroblasts/myofibroblasts, glial, epithelial, fat, vascular, smoothmuscle, and immune cells along with extracellular matrix (ECM) andextracellular molecules (Li Hanchen et al. Tumor Microenvironment: TheRole of the Tumor Stroma in Cancer. J of Cellular Biochemistry 101:805-815 (2007)).

Stromal modifying moieties described herein include moieties (e.g.,proteins, e.g., enzymes) capable of degrading a component of the stroma,e.g., an ECM component, e.g., a glycosaminoglycan, e.g., hyaluronan(also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin,dermatan sulfate, heparin sulfate, heparin, entactin, tenascin, aggrecanand keratin sulfate; or an extracellular protein, e.g., collagen,laminin, elastin, fibrinogen, fibronectin, and vitronectin.

Stromal Modifying Enzymes

In some embodiments, the stromal modifying moiety is an enzyme. Forexample, the stromal modifying moiety can include, but is not limited toa hyaluronidase, a collagenase, a chondroitinase, a matrixmetalloproteinase (e.g., macrophage metalloelastase).

Hyaluronidases

Hyaluronidases are a group of neutral- and acid-active enzymes foundthroughout the animal kingdom. Hyaluronidases vary with respect tosubstrate specificity, and mechanism of action. There are three generalclasses of hyaluronidases: (1) Mammalian-type hyaluronidases, (EC3.2.1.35) which are endo-beta-N-acetylhexosaminidases withtetrasaccharides and hexasaccharides as the major end products. Theyhave both hydrolytic and transglycosidase activities, and can degradehyaluronan and chondroitin sulfates; (2) Bacterial hyaluronidases (EC4.2.99.1) degrade hyaluronan and, and to various extents, chondroitinsulfate and dermatan sulfate. They are endo-beta-N-acetylhexosaminidasesthat operate by a beta elimination reaction that yields primarilydisaccharide end products; (3) Hyaluronidases (EC 3.2.1.36) fromleeches, other parasites, and crustaceans are endo-beta-glucuronidasesthat generate tetrasaccharide and hexasaccharide end products throughhydrolysis of the beta 1-3 linkage.

Mammalian hyaluronidases can be further divided into two groups: (1)neutral active and (2) acid active enzymes. There are sixhyaluronidase-like genes in the human genome, HYAL1, HYAL2, HYAL3 HYAL4HYALP1 and PH20/SPAM1. HYALP1 is a pseudogene, and HYAL3 has not beenshown to possess enzyme activity toward any known substrates. HYAL4 is achondroitinase and lacks activity towards hyaluronan. HYAL1 is theprototypical acid-active enzyme and PH20 is the prototypicalneutral-active enzyme. Acid active hyaluronidases, such as HYAL1 andHYAL2 lack catalytic activity at neutral pH. For example, HYAL1 has nocatalytic activity in vitro over pH 4.5 (Frost and Stern, “AMicrotiter-Based Assay for Hyaluronidase Activity Not RequiringSpecialized Reagents”, Analytical Biochemistry, vol. 251, pp. 263-269(1997). HYAL2 is an acid active enzyme with a very low specific activityin vitro.

In some embodiments the hyaluronidase is a mammalian hyaluronidase. Insome embodiments the hyaluronidase is a recombinant human hyaluronidase.In some embodiments, the hyaluronidase is a neutral activehyaluronidase. In some embodiments, the hyaluronidase is a neutralactive soluble hyaluronidase. In some embodiments, the hyaluronidase isa recombinant PH20 neutral-active enzyme. In some embodiments, thehyaluronidase is a recombinant PH20 neutral-active soluble enzyme. Insome embodiments the hyaluronidase is glycosylated. In some embodiments,the hyaluronidase possesses at least one N-linked glycan. A recombinanthyaluronidase can be produced using conventional methods known to thoseof skill in the art, e.g., U.S. Pat. No. 7,767,429, the entire contentsof which are incorporated by reference herein.

In some embodiments the hyaluronidase is rHuPH20 (also referred to asHylenex®; presently manufactured by Halozyme; approved by the FDA in2005 (see e.g., Scodeller P (2014) Hyaluronidase and other ExtracellularMatrix Degrading Enzymes for Cancer Therapy: New Uses andNano-Formulations. J Carcinog Mutage 5:178; U.S. Pat. Nos. 7,767,429;8,202,517; 7,431,380; 8,450,470; 8,772,246; 8,580,252, the entirecontents of each of which is incorporated by reference herein). rHuPH20is produced by genetically engineered CHO cells containing a DNA plasmidencoding for a soluble fragment of human hyaluronidase PH20. In someembodiments the hyaluronidase is glycosylated. In some embodiments, thehyaluronidase possesses at least one N-linked glycan. A recombinanthyaluronidase can be produced using conventional methods known to thoseof skill in the art, e.g., U.S. Pat. No. 7,767,429, the entire contentsof which are incorporated by reference herein. In some embodiments,rHuPH20 has a sequence at least 95% (e.g., at least 96%, 97%, 98%, 99%,100%) identical to the amino acid sequence of

(SEQ ID NO: 3306) LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS.

In any of the methods provided herein, the anti-hyaluronan agent can bean agent that degrades hyaluronan or can be an agent that inhibits thesynthesis of hyaluronan. For example, the anti-hyaluronan agent can be ahyaluronan degrading enzyme. In another example, the anti-hyaluronanagent or is an agent that inhibits hyaluronan synthesis. For example,the anti-hyaluronan agent is an agent that inhibits hyaluronan synthesissuch as a sense or antisense nucleic acid molecule against an HAsynthase or is a small molecule drug. For example, an anti-hyaluronanagent is 4-methylumbelliferone (MU) or a derivative thereof, orleflunomide or a derivative thereof. Such derivatives include, forexample, a derivative of 4-methylumbelliferone (MU) that is6,7-dihydroxy-4-methyl coumarin or 5,7-dihydroxy-4-methyl coumarin.

In further examples of the methods provided herein, the hyaluronandegrading enzyme is a hyaluronidase. In some examples, thehyaluronan-degrading enzyme is a PH20 hyaluronidase or truncated formthereof to lacking a C-terminal glycosylphosphatidylinositol (GPI)attachment site or a portion of the GPI attachment site. In specificexamples, the hyaluronidase is a PH20 selected from a human, monkey,bovine, ovine, rat, mouse or guinea pig PH20. For example, thehyaluronan-degrading enzyme is a human PH20 hyaluronidase that isneutral active and N-glycosylated and is selected from among (a) ahyaluronidase polypeptide that is a full-length PH20 or is a C-terminaltruncated form of the PH20, wherein the truncated form includes at leastamino acid residues 36-464 of SEQ ID NO: 139, such as 36-481, 36-482,36-483, where the full-length PH20 has the sequence of amino acids setforth in SEQ ID NO: 139; or (b) a hyaluronidase polypeptide comprising asequence of amino acids having at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitywith the polypeptide or truncated form of sequence of amino acids setforth in SEQ ID NO: 139; or (c) a hyaluronidase polypeptide of (a) or(b) comprising amino acid substitutions, whereby the hyaluronidasepolypeptide has a sequence of amino acids having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with the polypeptide set forth in SEQ ID NO: 139 orthe with the corresponding truncated forms thereof. In exemplaryexamples, the hyaluronan-degrading enzyme is a PH20 that comprises acomposition designated rHuPH20.

In other examples, the anti-hyaluronan agent is a hyaluronan degradingenzyme that is modified by conjugation to a polymer. The polymer can bea PEG and the anti-hyaluronan agent a PEGylated hyaluronan degradingenzyme. Hence, in some examples of the methods provided herein thehyaluronan-degrading enzyme is modified by conjugation to a polymer. Forexample, the hyaluronan-degrading enzyme is conjugated to a PEG, thusthe hyaluronan degrading enzyme is PEGylated. In an exemplary example,the hyaluronan-degrading enzyme is a PEGylated PH20 enzyme (PEGPH20). Inthe methods provided herein, the corticosteroid can be a glucocorticoidthat is selected from among cortisones, dexamethasones, hydrocortisones,methylprednisolones, prednisolones and prednisones.

Chondroitinases

Chondroitinases are enzymes found throughout the animal kingdom whichdegrade glycosaminoglycans, specifically chondroitins and chondroitinsulfates, through an endoglycosidase reaction. In some embodiments thechondroitinase is a mammalian chondroitinase. In some embodiments thechondroitinase is a recombinant human chondroitinase. In someembodiments the chondroitinase is HYAL4. Other exemplary chondroitinasesinclude chondroitinase ABC (derived from Proteus vulgaris; JapanesePatent Application Laid-open No 6-153947, T. Yamagata et al. J. Biol.Chem., 243, 1523 (1968), S. Suzuki et al, J. Biol. Chem., 243, 1543(1968)), chondroitinase AC (derived from Flavobacterium heparinum; T.Yamagata et al., J. Biol. Chem., 243, 1523 (1968)), chondroitinase AC II(derived from Arthrobacter aurescens; K. Hiyama, and S. Okada, J. Biol.Chem., 250, 1824 (1975), K. Hiyama and S. Okada, J. Biochem. (Tokyo),80, 1201 (1976)), Hyaluronidase ACIII (derived from Flavobacterium sp.Hp102; Hirofumi Miyazono et al., Seikagaku, 61, 1023 (1989)),chondroitinase B (derived from Flavobacterium heparinum; Y. M.Michelacci and C. P. Dietrich, Biochem. Biophys. Res. Commun., 56, 973(1974), Y. M. Michelacci and C. P. Dietrich, Biochem. J., 151, 121(1975), Kenichi Maeyama et al, Seikagaku, 57, 1189 (1985)),chondroitinase C (derived from Flavobacterium sp. Hp102; HirofumiMiyazono et al, Seikagaku, 61, 1023 (1939)), and the like.

Matrix Metalloproteinases

Matrix metalloproteases (MMPs) are zinc-dependent endopeptidases thatare the major proteases involved in extracellular matrix (ECM)degradation. MMPs are capable of degrading a wide range of extracellularmolecules and a number of bioactive molecules. Twenty-four MMP geneshave been identified in humans, which can be organized into six groupsbased on domain organization and substrate preference: Collagenases(MMP-1, -8 and -13), Gelatinases (MMP-2 and MMP-9), Stromelysins (MMP-3,-10 and -11), Matrilysin (MMP-7 and MMP-26), Membrane-type (MT)-MMPs(MMP-14, -15, -16, -17, -24 and -25) and others (MMP-12, -19, -20, -21,-23, -27 and -28). In some embodiments, the stromal modifying moiety isa human recombinant MMP (e.g., MMP-1, -2, -3, -4, -5, -6, -7, -8, -9,10, -11, -12, -13, -14, 15, -15, -17,-18, -19, 20, -21, -22, -23, or-24).

Collagenases

The three mammalian collagenases (MMP-1, -8, and -13) are the principalsecreted endopeptidases capable of cleaving collagenous extracellularmatrix. In addition to fibrillar collagens, collagenases can cleaveseveral other matrix and non-matrix proteins including growth factors.Collagenases are synthesized as inactive pro-forms, and once activated,their activity is inhibited by specific tissue inhibitors ofmetalloproteinases, TIMPs, as well as by non-specific proteinaseinhibitors (Ala-aho R et al. Biochimie. Collagenases in cancer. 2005March-April; 87(3-4):273-86). In some embodiments, the stromal modifyingmoiety is a collagenase. In some embodiments, the collagenase is a humanrecombinant collagenase. In some embodiments, the collagenase is MMP-1.In some embodiments, the collagenase is MMP-8. In some embodiments, thecollagenase is MMP-13.

Macrophage Metalloelastase

Macrophage metalloelastase (MME), also known as MMP-12, is a member ofthe stromelysin subgroup of MMPs and catalyzes the hydrolysis of solubleand insoluble elastin and a broad selection of matrix and nonmatrixsubstrates including type IV collagen, fibronectin, laminin,vitronectin, entactin, heparan, and chondroitin sulfates (Erja Kerkelaet al. Journal of Investigative Dermatology (2000) 114, 1113-1119;doi:10.1046/j.1523-1747.2000.00993). In some embodiments, the stromalmodifying moiety is a MME. In some embodiments, the MME is a humanrecombinant MME. In some embodiments, the MME is MMP-12.

Additional Stromal Modifying Moieties

In some embodiments, the stromal modifying moiety causes one or more of:decreases the level or production of a stromal or extracellular matrix(ECM) component; decreases tumor fibrosis; increases interstitial tumortransport; improves tumor perfusion; expands the tumor microvasculature;decreases interstitial fluid pressure (IFP) in a tumor; or decreases orenhances penetration or diffusion of an agent, e.g., a cancertherapeutic or a cellular therapy, into a tumor or tumor vasculature.

In some embodiments, the stromal or ECM component decreased is chosenfrom a glycosaminoglycan or an extracellular protein, or a combinationthereof. In some embodiments, the glycosaminoglycan is chosen fromhyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate,chondroitin, dermatan sulfate, heparin, heparin sulfate, entactin,tenascin, aggrecan and keratin sulfate. In some embodiments, theextracellular protein is chosen from collagen, laminin, elastin,fibrinogen, fibronectin, or vitronectin. In some embodiments, thestromal modifying moiety includes an enzyme molecule that degrades atumor stroma or extracellular matrix (ECM). In some embodiments, theenzyme molecule is chosen from a hyaluronidase molecule, a collagenasemolecule, a chondroitinase molecule, a matrix metalloproteinase molecule(e.g., macrophage metalloelastase), or a variant (e.g., a fragment) ofany of the aforesaid. The term “enzyme molecule” includes a full length,a fragment or a variant of the enzyme, e.g., an enzyme variant thatretains at least one functional property of the naturally-occurringenzyme.

In some embodiments, the stromal modifying moiety decreases the level orproduction of hyaluronic acid. In other embodiments, the stromalmodifying moiety comprises a hyaluronan degrading enzyme, an agent thatinhibits hyaluronan synthesis, or an antibody molecule againsthyaluronic acid.

In some embodiments, the hyaluronan degrading enzyme is a hyaluronidasemolecule, e.g., a full length or a variant (e.g., fragment thereof)thereof. In some embodiments, the hyaluronan degrading enzyme is activein neutral or acidic pH, e.g., pH of about 4-5. In some embodiments, thehyaluronidase molecule is a mammalian hyaluronidase molecule, e.g., arecombinant human hyaluronidase molecule, e.g., a full length or avariant (e.g., fragment thereof, e.g., a truncated form) thereof. Insome embodiments, the hyaluronidase molecule is chosen from HYAL1,HYAL2, or PH-20/SPAM1, or a variant thereof (e.g., a truncated formthereof). In some embodiments, the truncated form lacks a C-terminalglycosylphosphatidylinositol (GPI) attachment site or a portion of theGPI attachment site. In some embodiments, the hyaluronidase molecule isglycosylated, e.g., comprises at least one N-linked glycan.

In some embodiments, the hyaluronidase molecule comprises the amino acidsequence: LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS (SEQ ID NO:3311), or afragment thereof, or an amino acid sequence substantially identicalthereto (e.g., 95% to 99.9% identical thereto, or having at least oneamino acid alteration, but not more than five, ten or fifteenalterations (e.g., substitutions, deletions, or insertions, e.g.,conservative substitutions) to the amino acid sequence of SEQ ID NO:3311.

In some embodiments, the hyaluronidase molecule comprises:

(i) the amino acid sequence of 36-464 of SEQ ID NO: 3311;(ii) the amino acid sequence of 36-481, 36-482, or 36-483 of PH20,wherein PH20 has the sequence of amino acids set forth in SEQ ID NO:3311; or(iii) an amino acid sequence having at least 95% to 100% sequenceidentity to the polypeptide or truncated form of sequence of amino acidsset forth in SEQ ID NO: 3311; or(iv) an amino acid sequence having 30, 20, 10, 5 or fewer amino acidsubstitutions to the amino acid sequence set forth in SEQ ID NO: 3311.In some embodiments, the hyaluronidase molecule comprises an amino acidsequence at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, 100%)identical to the amino acid sequence of SEQ ID NO: 3311. In someembodiments, the hyaluronidase molecule is encoded by a nucleotidesequence at least 95% (e.g., at least 96%, 97%, 98%, 99%, 100%)identical to the nucleotide sequence of SEQ ID NO: 3311.

In some embodiments, the hyaluronidase molecule is PH20, e.g., rHuPH20.In some embodiments, the hyaluronidase molecule is HYAL1 and comprisesthe amino acid sequence:FRGPLLPNRPFTTVWNANTQWCLERHGVDVDVSVFDVVANPGQTFRGPDMTIFYSSQGTYPYYTPTGEPVFGGLPQNASLIAHLARTFQDILAAIPAPDFSGLAVIDWEAWRPRWAFNWDTKDIYRQRSRALVQAQHPDWPAPQVEAVAQDQFQGAARAWMAGTLQLGRALRPRGLWGFYGFPDCYNYDFLSPNYTGQCPSGIRAQNDQLGWLWGQSRALYPSIYMPAVLEGTGKSQMYVQHRVAEAFRVAVAAGDPNLPVLPYVQIFYDTTNHFLPLDELEHSLGESAAQGAAGVVLWVSWENTRTKESCQAIKEYMDTTLGPFILNVTSGALLCSQALCSGHGRCVRRTSHPKALLLLNPASFSIQLTPGGGPLSLRGALSLEDQAQMAVEFKCRCYPGWQAPWC ERKSMW (SEQID NO: 3312), or a fragment thereof, or an amino acid sequencesubstantially identical thereto (e.g., 95% to 99.9% identical thereto,or having at least one amino acid alteration, but not more than five,ten or fifteen alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions) to the amino acid sequenceof SEQ ID NO: 3312.

In some embodiments, the hyaluronan degrading enzyme, e.g., thehyaluronidase molecule, further comprises a polymer, e.g., is conjugatedto a polymer, e.g., PEG. In some embodiments, the hyaluronan-degradingenzyme is a PEGylated PH20 enzyme (PEGPH20). In some embodiments, thehyaluronan degrading enzyme, e.g., the hyaluronidase molecule, furthercomprises an immunoglobulin chain constant region (e.g., Fc region)chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3,and IgG4, more particularly, the heavy chain constant region of humanIgG1, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulinconstant region (e.g., the Fc region) is linked, e.g., covalently linkedto, the hyaluronan degrading enzyme, e.g., the hyaluronidase molecule.In some embodiments, the immunoglobulin chain constant region (e.g., Fcregion) is altered, e.g., mutated, to increase or decrease one or moreof: Fc receptor binding, antibody glycosylation, the number of cysteineresidues, effector cell function, or complement function. In someembodiments, the hyaluronan degrading enzyme, e.g., the hyaluronidasemolecule forms a dimer.

In some embodiments, the stromal modifying moiety comprises an inhibitorof the synthesis of hyaluronan, e.g., an HA synthase. In someembodiments, the inhibitor comprises a sense or an antisense nucleicacid molecule against an HA synthase or is a small molecule drug. Insome embodiments, the inhibitor is 4-methylumbelliferone (MU) or aderivative thereof (e.g., 6,7-dihydroxy-4-methyl coumarin or5,7-dihydroxy-4-methyl coumarin), or leflunomide or a derivativethereof.

In some embodiments, the stromal modifying moiety comprises antibodymolecule against hyaluronic acid.

In some embodiments, the stromal modifying moiety comprises acollagenase molecule, e.g., a mammalian collagenase molecule, or avariant (e.g., fragment) thereof. In some embodiments, the collagenasemolecule is collagenase molecule IV, e.g., comprising the amino acidsequence of:YNFFPRKPKWDKNQITYRIIGYTPDLDPETVDDAFARAFQVWSDVTPLRFSRIHDGEADIMINFGRWEHGDGYPFDGKDGLLAHAFAPGTGVGGDSHFDDDELWTLGEGQVVRVKYGNADGEYCKFPFLFNGKEYNSCTDTGRSDGFLWCSTTYNFEKDGKYGFCPHEALFTMGGNAEGQPCKFPFRFQGTSYDSCTTEGRTDGYRWCGTTEDYDRDKKYGFCPETAMSTVGGNSEGAPCVFPFTFLGNKYESCTSAGRSDGKMWCATTANYDDDRKWGFCPDQGYSLFLVAAHEFGHAMGLEHSQDPGALMAPIYTYTKNFRLSQDDIKGIQELYGASPDIDLGTGPTPTLGPVTPEICKQDIVFDGIAQIRGEIFFFKDRFIWRTVTPRDKPMGPLLVATFWPELPEKIDAVYEAPQEEKAVFFAGNEYWIYSASTLERGYPKPLTSLGLPPDVQRVDAAFNWSKNKKTYIFAGDKFWRYNEVKKKMDPGFPKLIADAWNAIPDNLDAVVDLQGGGHSYFFKGAYYLKLENQSLKSVKFGSIKSDWLGC (SEQ ID NO: 3313), or a fragment thereof, oran amino acid sequence substantially identical thereto (e.g., 95% to99.9% identical thereto, or having at least one amino acid alteration,but not more than five, ten or fifteen alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) to the aminoacid sequence of SEQ ID NO: 3313.

Linkers

The multispecific or multifunctional molecule disclosed herein canfurther include a linker, e.g., a linker between one or more of: theantigen binding domain and the cytokine molecule, the antigen bindingdomain and the immune cell engager, the antigen binding domain and thestromal modifying moiety, the cytokine molecule and the immune cellengager, the cytokine molecule and the stromal modifying moiety, theimmune cell engager and the stromal modifying moiety, the antigenbinding domain and the immunoglobulin chain constant region, thecytokine molecule and the immunoglobulin chain constant region, theimmune cell engager and the immunoglobulin chain constant region, or thestromal modifying moiety and the immunoglobulin chain constant region.In embodiments, the linker is chosen from: a cleavable linker, anon-cleavable linker, a peptide linker, a flexible linker, a rigidlinker, a helical linker, or a non-helical linker, or a combinationthereof.

In one embodiment, the multispecific molecule can include one, two,three or four linkers, e.g., a peptide linker. In one embodiment, thepeptide linker includes Gly and Ser. In some embodiments, the peptidelinker is selected from GGGGS (SEQ ID NO: 3307); GGGGSGGGGS (SEQ ID NO:3308); GGGGSGGGGSGGGGS (SEQ ID NO: 3309); and DVPSGPGGGGGSGGGGS (SEQ IDNO: 3310). In some embodiments, the peptide linker is a A(EAAAK)nA (SEQID NO: 3437) family of linkers (e.g., as described in Protein Eng.(2001) 14 (8): 529-532). These are stiff helical linkers with n rangingfrom 2-5. In some embodiments, the peptide linker is selected fromAEAAAKEAAAKAAA (SEQ ID NO: 3314); AEAAAKEAAAKEAAAKAAA (SEQ ID NO: 3315);AEAAAKEAAAKEAAAKEAAAKAAA (SEQ ID NO: 3316); andAEAAAKEAAAKEAAAKEAAAKEAAAKAAA (SEQ ID NO: 3317).

Nucleic Acids

Nucleic acids encoding the aforementioned antibody molecules, e.g.,anti-TCRβV antibody molecules, multispecific or multifunctionalmolecules are also disclosed.

In certain embodiments, the invention features nucleic acids comprisingnucleotide sequences that encode heavy and light chain variable regionsand CDRs or hypervariable loops of the antibody molecules, as describedherein. For example, the invention features a first and second nucleicacid encoding heavy and light chain variable regions, respectively, ofan antibody molecule chosen from one or more of the antibody moleculesdisclosed herein. The nucleic acid can comprise a nucleotide sequence asset forth in the tables herein, or a sequence substantially identicalthereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or moreidentical thereto, or which differs by no more than 3, 6, 15, 30, or 45nucleotides from the sequences shown in the tables herein.

In certain embodiments, the nucleic acid can comprise a nucleotidesequence encoding at least one, two, or three CDRs or hypervariableloops from a heavy chain variable region having an amino acid sequenceas set forth in the tables herein, or a sequence substantiallyhomologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99%or more identical thereto, and/or having one or more substitutions,e.g., conserved substitutions). In other embodiments, the nucleic acidcan comprise a nucleotide sequence encoding at least one, two, or threeCDRs or hypervariable loops from a light chain variable region having anamino acid sequence as set forth in the tables herein, or a sequencesubstantially homologous thereto (e.g., a sequence at least about 85%,90%, 95%, 99% or more identical thereto, and/or having one or moresubstitutions, e.g., conserved substitutions). In yet anotherembodiment, the nucleic acid can comprise a nucleotide sequence encodingat least one, two, three, four, five, or six CDRs or hypervariable loopsfrom heavy and light chain variable regions having an amino acidsequence as set forth in the tables herein, or a sequence substantiallyhomologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99%or more identical thereto, and/or having one or more substitutions,e.g., conserved substitutions).

In certain embodiments, the nucleic acid can comprise a nucleotidesequence encoding at least one, two, or three CDRs or hypervariableloops from a heavy chain variable region having the nucleotide sequenceas set forth in the tables herein, a sequence substantially homologousthereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or moreidentical thereto, and/or capable of hybridizing under the stringencyconditions described herein). In another embodiment, the nucleic acidcan comprise a nucleotide sequence encoding at least one, two, or threeCDRs or hypervariable loops from a light chain variable region havingthe nucleotide sequence as set forth in the tables herein, or a sequencesubstantially homologous thereto (e.g., a sequence at least about 85%,90%, 95%, 99% or more identical thereto, and/or capable of hybridizingunder the stringency conditions described herein). In yet anotherembodiment, the nucleic acid can comprise a nucleotide sequence encodingat least one, two, three, four, five, or six CDRs or hypervariable loopsfrom heavy and light chain variable regions having the nucleotidesequence as set forth in the tables herein, or a sequence substantiallyhomologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99%or more identical thereto, and/or capable of hybridizing under thestringency conditions described herein).

In certain embodiments, the nucleic acid can comprise a nucleotidesequence encoding a cytokine molecule, an immune cell engager, or astromal modifying moiety disclosed herein.

In another aspect, the application features host cells and vectorscontaining the nucleic acids described herein. The nucleic acids may bepresent in a single vector or separate vectors present in the same hostcell or separate host cell, as described in more detail hereinbelow.

Vectors

Further provided herein are vectors comprising the nucleotide sequencesencoding antibody molecules, e.g., anti-TCRβV antibody molecules, or amultispecific or multifunctional molecule described herein. In oneembodiment, the vectors comprise nucleic acid sequences encodingantibody molecules, e.g., anti-TCRβV antibody molecules, ormultispecific or multifunctional molecule described herein. In oneembodiment, the vectors comprise the nucleotide sequences describedherein. The vectors include, but are not limited to, a virus, plasmid,cosmid, lambda phage or a yeast artificial chromosome (YAC).

Numerous vector systems can be employed. For example, one class ofvectors utilizes DNA elements which are derived from animal viruses suchas, for example, bovine papilloma virus, polyoma virus, adenovirus,vaccinia virus, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV orMOMLV) or SV40 virus. Another class of vectors utilizes RNA elementsderived from RNA viruses such as Semliki Forest virus, Eastern EquineEncephalitis virus and Flaviviruses.

Additionally, cells which have stably integrated the DNA into theirchromosomes may be selected by introducing one or more markers whichallow for the selection of transfected host cells. The marker mayprovide, for example, prototropy to an auxotrophic host, biocideresistance (e.g., antibiotics), or resistance to heavy metals such ascopper, or the like. The selectable marker gene can be either directlylinked to the DNA sequences to be expressed, or introduced into the samecell by cotransformation. Additional elements may also be needed foroptimal synthesis of mRNA. These elements may include splice signals, aswell as transcriptional promoters, enhancers, and termination signals.

Once the expression vector or DNA sequence containing the constructs hasbeen prepared for expression, the expression vectors may be transfectedor introduced into an appropriate host cell. Various techniques may beemployed to achieve this, such as, for example, protoplast fusion,calcium phosphate precipitation, electroporation, retroviraltransduction, viral transfection, gene gun, lipid based transfection orother conventional techniques. In the case of protoplast fusion, thecells are grown in media and screened for the appropriate activity.

Methods and conditions for culturing the resulting transfected cells andfor recovering the antibody molecule produced are known to those skilledin the art, and may be varied or optimized depending upon the specificexpression vector and mammalian host cell employed, based upon thepresent description.

Cells

In another aspect, the application features host cells and vectorscontaining the nucleic acids described herein. The nucleic acids may bepresent in a single vector or separate vectors present in the same hostcell or separate host cell. The host cell can be a eukaryotic cell,e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryoticcell, e.g., E. coli. For example, the mammalian cell can be a culturedcell or a cell line. Exemplary mammalian cells include lymphocytic celllines (e.g., NSO), Chinese hamster ovary cells (CHO), COS cells, oocytecells, and cells from a transgenic animal, e.g., mammary epithelialcell.

The invention also provides host cells comprising a nucleic acidencoding an antibody molecule as described herein.

In one embodiment, the host cells are genetically engineered to comprisenucleic acids encoding the antibody molecule.

In one embodiment, the host cells are genetically engineered by using anexpression cassette. The phrase “expression cassette,” refers tonucleotide sequences, which are capable of affecting expression of agene in hosts compatible with such sequences. Such cassettes may includea promoter, an open reading frame with or without introns, and atermination signal. Additional factors necessary or helpful in effectingexpression may also be used, such as, for example, an induciblepromoter.

The invention also provides host cells comprising the vectors describedherein.

The cell can be, but is not limited to, a eukaryotic cell, a bacterialcell, an insect cell, or a human cell. Suitable eukaryotic cellsinclude, but are not limited to, Vero cells, HeLa cells, COS cells, CHOcells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cellsinclude, but are not limited to, Sf9 cells.

Method of Expanding Cells with Anti-TCRVB Antibodies

Any of the compositions and methods described herein can be used toexpand an immune cell population. An immune cell provided hereinincludes an immune cell derived from a hematopoietic stem cell or animmune cell derived from a non-hematopoietic stem cell, e.g., bydifferentiation or de-differentiation.

An immune cell includes a hematopoietic stem cell, progeny thereofand/or cells that have differentiated from said HSC, e.g., lymphoidcells or myeloid cells. An immune cell can be an adaptive immune cell oran innate immune cell. Examples of immune cells include T cells, Bcells, Natural Killer cells, Natural Killer T cells, neutrophils,dendritic cells, monocytes, macrophages, and granulocytes.

In some embodiments of any of the methods of compositions disclosedherein, an immune cell is a T cell. In some embodiments, a T cellincludes a CD4+ T cell, a CD8+ T cell, a TCR alpha-beta T cell, a TCRgamma-delta T cell. In some embodiments, a T cell comprises a memory Tcell (e.g., a central memory T cell, or an effector memory T cell (e.g.,a TEMRA) or an effector T cell. In some embodiments, a T cell comprisesa tumor infiltrating lymphocyte (TIL).

In some embodiments of any of the methods of compositions disclosedherein, an immune cell is an NK cell.

In some embodiments of any of the methods of compositions disclosedherein, an immune cell is a TIL. TILs are immune cells (e.g., T cells, Bcells or NK cells) that can be found in a tumor or around a tumor (e.g.,in the stroma or tumor microenvironment of a tumor), e.g., a solidtumor, e.g., as described herein. TILs can be obtained from a samplefrom a subject having cancer, e.g., a biopsy or a surgical sample. Insome embodiments, TILs can be expanded using a method disclosed herein.In some embodiments, a population of expanded TILs, e.g., expanded usinga method disclosed herein, can be administered to a subject to treat adisease, e.g., a cancer.

In certain aspects of the present disclosure, immune cells, e.g., Tcells (e.g., TILs), can be obtained from a unit of blood collected froma subject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In one aspect, cells from the circulatingblood of an individual are obtained by apheresis. The apheresis producttypically contains lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and platelets. In one aspect, the cells collected by apheresismay be washed to remove the plasma fraction and, optionally, to placethe cells in an appropriate buffer or media for subsequent processingsteps. In one embodiment, the cells are washed with phosphate bufferedsaline (PBS). In an alternative embodiment, the wash solution lackscalcium and may lack magnesium or may lack many if not all divalentcations. The methods described herein can include more than oneselection step, e.g., more than one depletion step.

In one embodiment, the methods of the application can utilize culturemedia conditions comprising DMEM, DMEM F12, RPMI 1640, and/or AIM Vmedia. The media can be supplemented with glutamine, HEPES buffer (e.g.,10 mM), serum (e.g., heat-inactivated serum, e.g., 10%), and/or betamercaptoethanol (e.g., 55 uM). IN some embodiments, the cultureconditions disclosed herein comprise one or more supplements, cytokines,growth factors, or hormones. In some embodiments, the culture conditioncomprises one or more of IL-2, IL-15, or IL-7, or a combination thereof.

Immune effector cells such as T cells may be activated and expandedgenerally using methods as described, for example, in U.S. Pat. Nos.6,352,694; 6,534,055; or 6,905,680. Generally, a population of immunecells, may be expanded by contact with an agent that stimulates aCD3/TCR complex associated signal and a ligand that stimulates acostimulatory molecule on the surface of the T cells; and/or by contactwith a cytokine, e.g., IL-2, IL-15 or IL-7. T cell expansion protocolscan also include stimulation, such as by contact with an anti-CD3antibody, or antigen-binding fragment thereof, or an anti-CD2 antibodyimmobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For example, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and ananti-CD28 antibody can be used. Examples of an anti-CD28 antibodyinclude 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used ascan other methods commonly known in the art (Berg et al., TransplantProc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63,1999).

A TIL population can also be expanded by methods known in the art. Forexample, a population of TILs can be expanded as described in Hall etal., Journal for ImmunoTherapy of Cancer (2016) 4:61, the entirecontents of which are hereby incorporated by reference. Briefly, TILscan be isolated from a sample by mechanical and/or physical digestion.The resultant TIL population can be stimulated with an anti-CD3 antibodyin the presence of non-dividing feeder cells. In some embodiments, theTIL population can be cultured, e.g., expanded, in the presence of IL-2,e.g., human IL-2. In some embodiments, the TIL cells can be cultured,e.g., expanded fora period of at least 1-21 days, e.g., at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21days.

As disclosed herein, in some embodiments, an immune cell population(e.g., a T cell (e.g., a T_(EMRA) cell or a TIL population) can beexpanded by contacting the immune cell population with an anti-TCRVBantibody, e.g., as described herein.

In some embodiments, the expansion occurs in vivo, e.g., in a subject.In some embodiments, a subject is administered an anti-TCRβV antibodymolecule disclosed herein resulting in expansion of immune cells invivo.

In some embodiments, the expansion occurs ex vivo, e.g., in vitro. Insome embodiments, cells from a subject, e.g., T cells, e.g., TIL cells,are expanded in vitro with an anti-TCRβV antibody molecule disclosedherein. In some embodiments, the expanded TILs are administered to thesubject to treat a disease or a symptom of a disease.

In some embodiments, a method of expansion disclosed herein results inan expansion of at least 1.1-10 fold, 10-20 fold, or 20-50 foldexpansion. In some embodiments, the expansion is at least 1.1, 1.2, 1.3,1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50fold expansion.

In some embodiments, a method of expansion disclosed herein comprisesculturing, e.g., expanding, the cells for at least about 4 hours, 6hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, or 22 hours. Insome embodiments, a method of expansion disclosed herein comprisesculturing, e.g., expanding, the cells for at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days. In someembodiments, a method of expansion disclosed herein comprises culturing,e.g., expanding, the cells for at least about 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks.

In some embodiments, a method of expansion disclosed herein is performedon immune cells obtained from a healthy subject.

In some embodiments, a method of expansion disclosed herein is performedon immune cells (e.g., TILs) obtained from a subject having a disease,e.g., a cancer, e.g., a solid tumor as disclosed herein.

In some embodiments, a method of expansion disclosed herein furthercomprises contacting the population of cells with an agent, thatpromotes, e.g., increases, immune cell expansion. In some embodiments,the agent comprises an immune checkpoint inhibitor, e.g., a PD-1inhibitor, a LAG-3 inhibitor, a CTLA4 inhibitor, or a TIM-3 inhibitor.In some embodiments, the agent comprises a 4-1BB agonist, e.g., ananti-4-1BB antibody.

Without wishing to be bound by theory, it is believed that an anti-TCRβVantibody molecule disclosed herein can expand, e.g., selectively orpreferentially expand, T cells expressing a T cell receptor (TCR)comprising a TCR alpha and/or TCR beta molecule, e.g., TCR alpha-beta Tcells (αβ T cells). In some embodiments, an anti-TCRβV antibody moleculedisclosed herein does not expand, or induce proliferation of T cellsexpressing a TCR comprising a TCR gamma and/or TCR delta molecule, e.g.,TCR gamma-delta T cells (γδ T cells). In some embodiments, an anti-TCRβVantibody molecule disclosed herein, selectively or preferentiallyexpands αβ T cells over γδ T cells.

Without wishing to be bound by theory, it is believed that, in someembodiments, γδ T cells are associated with cytokine release syndrome(CRS) and/or neurotoxicity (NT). In some embodiments, an anti-TCRβVantibody molecule disclosed herein results in selective expansion ofnon-γδ T cells, e.g., expansion of αβ T cells, thus reducing CRS and/orNT.

In some embodiments, any of the compositions or methods disclosed hereinresult in an immune cell population having a reduction of, e.g.,depletion of, γδ T cells. In some embodiments, the immune cellpopulation is contacted with an agent that reduces, e.g., inhibits ordepletes, γδ T cells, e.g., an anti-IL-17 antibody or an agent thatbinds to a TCR gamma and/or TCR delta molecule.

Uses and Combination Therapies

Methods described herein include treating a cancer in a subject by usingan anti-TCRβV antibody molecule, a multispecific or multifunctionalmolecule described herein, e.g., using a pharmaceutical compositiondescribed herein. Also provided are methods for reducing or amelioratinga symptom of a cancer in a subject, as well as methods for inhibitingthe growth of a cancer and/or killing one or more cancer cells. Inembodiments, the methods described herein decrease the size of a tumorand/or decrease the number of cancer cells in a subject administeredwith a described herein or a pharmaceutical composition describedherein.

In embodiments, the cancer is a hematological cancer. In embodiments,the hematological cancer is a leukemia or a lymphoma. As used herein, a“hematologic cancer” refers to a tumor of the hematopoietic or lymphoidtissues, e.g., a tumor that affects blood, bone marrow, or lymph nodes.Exemplary hematologic malignancies include, but are not limited to,leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenousleukemia (CML), hairy cell leukemia, acute monocytic leukemia (AMoL),chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia(JMML), or large granular lymphocytic leukemia), lymphoma (e.g.,AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma(e.g., classical Hodgkin lymphoma or nodular lymphocyte-predominantHodgkin lymphoma), mycosis fungoides, non-Hodgkin lymphoma (e.g., B-cellnon-Hodgkin lymphoma (e.g., Burkitt lymphoma, small lymphocytic lymphoma(CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma,immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,or mantle cell lymphoma) or T-cell non-Hodgkin lymphoma (mycosisfungoides, anaplastic large cell lymphoma, or precursor T-lymphoblasticlymphoma)), primary central nervous system lymphoma, Sezary syndrome,Waldenström macroglobulinemia), chronic myeloproliferative neoplasm,Langerhans cell histiocytosis, multiple myeloma/plasma cell neoplasm,myelodysplastic syndrome, or myelodysplastic/myeloproliferativeneoplasm.

In embodiments, the cancer is a myeloproliferative neoplasm, e.g.,primary or idiopathic myelofibrosis (MF), essential thrombocytosis (ET),polycythemia vera (PV), or chronic myelogenous leukemia (CIVIL). Inembodiments, the cancer is myelofibrosis. In embodiments, the subjecthas myelofibrosis. In embodiments, the subject has a calreticulinmutation, e.g., a calreticulin mutation disclosed herein. Inembodiments, the subject does not have the JAK2-V617F mutation. Inembodiments, the subject has the JAK2-V617F mutation. In embodiments,the subject has a MPL mutation. In embodiments, the subject does nothave a MPL mutation.

In embodiments, the cancer is a solid cancer. Exemplary solid cancersinclude, but are not limited to, ovarian cancer, rectal cancer, stomachcancer, testicular cancer, cancer of the anal region, uterine cancer,colon cancer, rectal cancer, renal-cell carcinoma, liver cancer,non-small cell carcinoma of the lung, cancer of the small intestine,cancer of the esophagus, melanoma, Kaposi's sarcoma, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, bone cancer, pancreatic cancer, skincancer, cancer of the head or neck, cutaneous or intraocular malignantmelanoma, uterine cancer, brain stem glioma, pituitary adenoma,epidermoid cancer, carcinoma of the cervix squamous cell cancer,carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the vagina, sarcoma of soft tissue, cancer of the urethra,carcinoma of the vulva, cancer of the penis, cancer of the bladder,cancer of the kidney or ureter, carcinoma of the renal pelvis, spinalaxis tumor, neoplasm of the central nervous system (CNS), primary CNSlymphoma, tumor angiogenesis, metastatic lesions of said cancers, orcombinations thereof.

In embodiments, the anti-TCRβV antibody molecule, multispecific ormultifunctional molecules (or pharmaceutical composition) areadministered in a manner appropriate to the disease to be treated orprevented. The quantity and frequency of administration will bedetermined by such factors as the condition of the patient, and the typeand severity of the patient's disease. Appropriate dosages may bedetermined by clinical trials. For example, when “an effective amount”or “a therapeutic amount” is indicated, the precise amount of thepharmaceutical composition (or multispecific or multifunctionalmolecules) to be administered can be determined by a physician withconsideration of individual differences in tumor size, extent ofinfection or metastasis, age, weight, and condition of the subject. Inembodiments, the pharmaceutical composition described herein can beadministered at a dosage of 10⁴ to 10⁹ cells/kg body weight, e.g., 10⁵to 10⁶ cells/kg body weight, including all integer values within thoseranges. In embodiments, the pharmaceutical composition described hereincan be administered multiple times at these dosages. In embodiments, thepharmaceutical composition described herein can be administered usinginfusion techniques described in immunotherapy (see, e.g., Rosenberg etal., New Eng. J. of Med. 319:1676, 1988).

In embodiments, the anti-TCRβV antibody molecule, multispecific ormultifunctional molecules or pharmaceutical composition is administeredto the subject parentally. In embodiments, the cells are administered tothe subject intravenously, subcutaneously, intratumorally, intranodally,intramuscularly, intradermally, or intraperitoneally. In embodiments,the cells are administered, e.g., injected, directly into a tumor orlymph node. In embodiments, the cells are administered as an infusion(e.g., as described in Rosenberg et al., New Eng. J. of Med. 319:1676,1988) or an intravenous push. In embodiments, the cells are administeredas an injectable depot formulation.

In embodiments, the subject is a mammal. In embodiments, the subject isa human, monkey, pig, dog, cat, cow, sheep, goat, rabbit, rat, or mouse.In embodiments, the subject is a human. In embodiments, the subject is apediatric subject, e.g., less than 18 years of age, e.g., less than 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less years ofage. In embodiments, the subject is an adult, e.g., at least 18 years ofage, e.g., at least 19, 20, 21, 22, 23, 24, 25, 25-30, 30-35, 35-40,40-50, 50-60, 60-70, 70-80, or 80-90 years of age.

Methods of Cancer Treatment

Methods described herein include treating a cancer in a subject by usingan anti-TCRβV antibody molecule, e.g., using a pharmaceuticalcomposition described herein. Also provided are methods for reducing orameliorating a symptom of a cancer in a subject, as well as methods forinhibiting the growth of a cancer and/or killing one or more cancercells. In embodiments, the methods described herein decrease the size ofa tumor and/or decrease the number of cancer cells in a subjectadministered with a described herein or a pharmaceutical compositiondescribed herein.

Disclosed herein are methods of treating a subject having a cancercomprising acquiring a status of one or more TCRBV molecules in asubject. In some embodiments, a higher, e.g., increased, level oractivity of one or more TCRBV molecules in a subject, e.g., in a samplefrom a subject, is indicative of a bias, e.g., a preferential expansion,e.g., clonal expansion, of T cells expressing said one or more TCRβVmolecules in the subject.

Without wishing to be bound by theory, it is believed that a biased Tcell population, e.g., a T cell population expressing a TCRBV molecule,is antigen-specific for a disease antigen, e.g., a cancer antigen (WangC Y, et al., Int J Oncol. (2016) 48(6):2247-56). In some embodiments,the cancer antigen comprises a cancer associated antigen or aneoantigen. In some embodiments, a subject having a cancer, e.g., asdisclosed herein, has a higher, e.g., increased, level or activity ofone or more TCRβV molecules associated with the cancer. In someembodiments, the TCRβV molecule is associated with, e.g., recognizes, acancer antigen, e.g., a cancer associated antigen or a neoantigen.

Accordingly, disclosed herein are methods of expanding an immuneeffector cell population obtained from a subject, comprising acquiring astatus of one or more TCRβV molecules in a sample from the subject,comprising contacting said immune effector cell population with ananti-TCRβV antibody molecule disclosed herein, e.g., an anti-TCRβVantibody molecule that binds to the same TCRβV molecule that is higher,e.g., increased in the immune effector cell population in the samplefrom the subject. In some embodiments, contacting the population ofimmune effector cells (e.g., comprising T cells that express one or moreTCRβV molecules) with an anti-TCRβV molecule results in expansion of thepopulation of immune effector cells expressing one or more TCRβVmolecules. In some embodiments, the expanded population, or a portionthereof, is administered to the subject (e.g., same subject from whomthe immune effector cell population was obtained), to treat the cancer.In some embodiments, the expanded population, or a portion thereof, isadministered to a different subject (e.g., not the same subject fromwhom the immune effector cell population was obtained), to treat thecancer.

Also disclosed herein, are methods of treating a subject having acancer, comprising: acquiring a status of one or more TCRβV molecules ina sample from the subject, and determining whether the one or more TCRβVmolecules is higher, e.g., increased, in a sample from the subjectcompared to a reference value, wherein responsive to said determination,administering to the subject an effective amount of an anti-TCRβVantibody molecule, e.g., an agonistic anti-TCRβV antibody molecule,e.g., as described herein.

In some embodiments of any of the methods or composition for usedisclosed herein, the subject has B-CLL. In some embodiments, a subjecthaving B-CLL has a higher, e.g., increased, level or activity of one ormore TCRβV molecules, e.g., one or more TCRβV molecules comprising: (i)TCRβ V6 subfamily comprising, e.g., TCRβ V6-4*01, TCRβ V6-4*02, TCRβV6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβV6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01; (ii) TCRβ V5 subfamily comprisingTCRβ V5-6*01, TCRβ V5-4*01, or TCRβ V5-8*01; (iii) TCRβ V3 subfamilycomprising TCRβ V3-1*01; (iv) TCRβ V2 subfamily comprising TCRβ V2*01;or (v) TCRβ V19 subfamily comprising TCRβ V19*01, or TCRβ V19*02.

In some embodiments, a subject having B-CLL has a higher, e.g.,increased, level or activity of a TCRβ V6 subfamily comprising, e.g.,TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01,TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01.In some embodiments, the subject is administered an anti-TCRβV molecule(e.g., an agonistic anti-TCRβV molecule as described herein) that bindsto one or more members of the TCRβ V6 subfamily. In some embodiments,administration of the an anti-TCRβV molecule results in expansion ofimmune cells expressing one or more members of the TCRβ V6 subfamily.

In some embodiments, a subject having B-CLL has a higher, e.g.,increased, level or activity of a TCRβ V5 subfamily comprising TCRβV5-6*01, TCRβ V5-4*01, or TCRβ V5-8*01. In some embodiments, the subjectis administered an anti-TCRβV molecule (e.g., an agonistic anti-TCRβVmolecule as described herein) that binds to one or more members of theTCRβ V5 subfamily. In some embodiments, administration of the ananti-TCRβV molecule results in expansion of immune cells expressing oneor more members of the TCRβ V5 subfamily.

In some embodiments, a subject having B-CLL has a higher, e.g.,increased, level or activity of a TCRβ V3 subfamily comprising TCRβV3-1*01. In some embodiments, the subject is administered an anti-TCRβVmolecule (e.g., an agonistic anti-TCRβV molecule as described herein)that binds to one or more members of the TCRβ V3 subfamily. In someembodiments, administration of the an anti-TCRβV molecule results inexpansion of immune cells expressing one or more members of the TCRβ V3subfamily.

In some embodiments, a subject having B-CLL has a higher, e.g.,increased, level or activity of a TCRβ V2 subfamily comprising TCRβV2*01. In some embodiments, the subject is administered an anti-TCRβVmolecule (e.g., an agonistic anti-TCRβV molecule as described herein)that binds to one or more members of the TCRβ V2 subfamily. In someembodiments, administration of the an anti-TCRβV molecule results inexpansion of immune cells expressing one or more members of the TCRβ V2subfamily.

In some embodiments, a subject having B-CLL has a higher, e.g.,increased, level or activity of a TCRβ V19 subfamily comprising TCRβV19*01, or TCRβ V19*02. In some embodiments, the subject is administeredan anti-TCRβV molecule (e.g., an agonistic anti-TCRBV molecule asdescribed herein) that binds to one or more members of the TCRβ V19subfamily. In some embodiments, administration of the an anti-TCRβVmolecule results in expansion of immune cells expressing one or moremembers of the TCRβ V19 subfamily.

In some embodiments of any of the methods or composition for usedisclosed herein, the subject has melanoma. In some embodiments, asubject having melanoma has a higher, e.g., increased, level or activityof one or more TCRβV molecules, e.g., one or more TCRβV moleculescomprising the TCRβ V6 subfamily comprising, e.g., TCRβ V6-4*01, TCRβV6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβV6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01. In someembodiments, the subject is administered an anti-TCRβV molecule (e.g.,an agonistic anti-TCRβV molecule as described herein) that binds to oneor more members of the TCRβ V6 subfamily. In some embodiments,administration of the an anti-TCRβV molecule results in expansion ofimmune cells expressing one or more members of the TCRβ V6 subfamily.

In some embodiments of any of the methods or composition for usedisclosed herein, the subject has DLBCL. In some embodiments, a subjecthaving melanoma has a higher, e.g., increased, level or activity of oneor more TCRβV molecules, e.g., one or more TCRβV molecules comprising:(i) TCRβ V13 subfamily comprising TCRβ V13*01; (ii) TCRβ V3 subfamilycomprising TCRβ V3-1*01; or (iii) TCRβ V23 subfamily.

In some embodiments, a subject having DLBCL has a higher, e.g.,increased, level or activity of a TCRβ V13 subfamily comprising TCRβV13*01. In some embodiments, the subject is administered an anti-TCRβVmolecule (e.g., an agonistic anti-TCRβV molecule as described herein)that binds to one or more members of the TCRβ V13 subfamily. In someembodiments, administration of the an anti-TCRβV molecule results inexpansion of immune cells expressing one or more members of the TCRβ V13subfamily.

In some embodiments, a subject having DLBCL has a higher, e.g.,increased, level or activity of a TCRβ V3 subfamily comprising TCRβV3-1*01. In some embodiments, the subject is administered an anti-TCRβVmolecule (e.g., an agonistic anti-TCRβV molecule as described herein)that binds to one or more members of the TCRβ V3 subfamily. In someembodiments, administration of the an anti-TCRβV molecule results inexpansion of immune cells expressing one or more members of the TCRβ V3subfamily.

In some embodiments, a subject having DLBCL has a higher, e.g.,increased, level or activity of a TCRβ V23 subfamily. In someembodiments, the subject is administered an anti-TCRβV molecule (e.g.,an agonistic anti-TCRβV molecule as described herein) that binds to oneor more members of the TCRβ V23 subfamily. In some embodiments,administration of the an anti-TCRβV molecule results in expansion ofimmune cells expressing one or more members of the TCRβ V23 subfamily.

In some embodiments of any of the methods or composition for usedisclosed herein, the subject has CRC. In some embodiments, a subjecthaving melanoma has a higher, e.g., increased, level or activity of oneor more TCRβV molecules, e.g., one or more TCRβV molecules comprising:(i) TCRβ V19 subfamily comprising TCRβ V19*01, or TCRβ V19*02; (ii) TCRβV12 subfamily comprising TCRβ V12-4*01, TCRβ V12-3*01, or TCRβ V12-5*01;(iii) TCRβ V16 subfamily comprising TCRβ V16*01; or (iv) TCRβ V21subfamily.

In some embodiments, a subject having CRC has a higher, e.g., increased,level or activity of a TCRβ V19 subfamily comprising TCRβ V19*01, orTCRβ V19*02. In some embodiments, the subject is administered ananti-TCRβV molecule (e.g., an agonistic anti-TCRβV molecule as describedherein) that binds to one or more members of the TCRβ V19 subfamily. Insome embodiments, administration of the an anti-TCRβV molecule resultsin expansion of immune cells expressing one or more members of the TCRβV19 subfamily.

In some embodiments, a subject having CRC has a higher, e.g., increased,level or activity of a TCRβ V12 subfamily comprising TCRβ V12-4*01, TCRβV12-3*01, or TCRβ V12-5*01. In some embodiments, the subject isadministered an anti-TCRβV molecule (e.g., an agonistic anti-TCRβVmolecule as described herein) that binds to one or more members of theTCRβ V12 subfamily. In some embodiments, administration of the ananti-TCRβV molecule results in expansion of immune cells expressing oneor more members of the TCRβ V12 subfamily.

In some embodiments, a subject having CRC has a higher, e.g., increased,level or activity of a TCRβ V16 subfamily comprising TCRβ V16*01. Insome embodiments, the subject is administered an anti-TCRβV molecule(e.g., an agonistic anti-TCRβV molecule as described herein) that bindsto one or more members of the TCRβ V16 subfamily. In some embodiments,administration of the an anti-TCRβV molecule results in expansion ofimmune cells expressing one or more members of the TCRβ V16 subfamily.

In some embodiments, a subject having CRC has a higher, e.g., increased,level or activity of a TCRβ V21 subfamily. In some embodiments, thesubject is administered an anti-TCRβV molecule (e.g., an agonisticanti-TCRβV molecule as described herein) that binds to one or moremembers of the TCRβ V21 subfamily. In some embodiments, administrationof the an anti-TCRβV molecule results in expansion of immune cellsexpressing one or more members of the TCRβ V21 subfamily.

In some embodiments, acquiring a value for the status, e.g., presence,level and/or activity, of one or more TCRβV molecules comprisesacquiring a measure of the T cell receptor (TCR) repertoire of a sample.In some embodiments, the value comprises a measure of the clonotype of apopulation of T cells in the sample.

In some embodiments, a value for the status of one or more TCRβVmolecules is obtained, e.g., measured, using an assay described in WangC Y, et al., Int J Oncol. (2016) 48(6):2247-56, the entire contents ofwhich are hereby incorporated by reference.

In some embodiments, a value for the status of one or more TCRβVmolecules is obtained, e.g., measured, using flow cytometry.

Combination Therapies

The anti-TCRβV antibody molecule, multispecific or multifunctionalmolecules disclosed herein can be used in combination with a secondtherapeutic agent or procedure.

In embodiments, the anti-TCRβV antibody molecule, multispecific ormultifunctional molecule and the second therapeutic agent or procedureare administered/performed after a subject has been diagnosed with acancer, e.g., before the cancer has been eliminated from the subject. Inembodiments, the anti-TCRβV antibody molecule, multispecific ormultifunctional molecule and the second therapeutic agent or procedureare administered/performed simultaneously or concurrently. For example,the delivery of one treatment is still occurring when the delivery ofthe second commences, e.g., there is an overlap in administration of thetreatments. In other embodiments, the anti-TCRβV antibody molecule,multispecific or multifunctional molecule and the second therapeuticagent or procedure are administered/performed sequentially. For example,the delivery of one treatment ceases before the delivery of the othertreatment begins.

In embodiments, combination therapy can lead to more effective treatmentthan monotherapy with either agent alone. In embodiments, thecombination of the first and second treatment is more effective (e.g.,leads to a greater reduction in symptoms and/or cancer cells) than thefirst or second treatment alone. In embodiments, the combination therapypermits use of a lower dose of the first or the second treatmentcompared to the dose of the first or second treatment normally requiredto achieve similar effects when administered as a monotherapy. Inembodiments, the combination therapy has a partially additive effect,wholly additive effect, or greater than additive effect.

In one embodiment, the anti-TCRBV antibody, multispecific ormultifunctional molecule is administered in combination with a therapy,e.g., a cancer therapy (e.g., one or more of anti-cancer agents,immunotherapy, photodynamic therapy (PDT), surgery and/or radiation).The terms “chemotherapeutic,” “chemotherapeutic agent,” and “anti-canceragent” are used interchangeably herein. The administration of themultispecific or multifunctional molecule and the therapy, e.g., thecancer therapy, can be sequential (with or without overlap) orsimultaneous. Administration of the anti-TCRBV antibody, multispecificor multifunctional molecule can be continuous or intermittent during thecourse of therapy (e.g., cancer therapy). Certain therapies describedherein can be used to treat cancers and non-cancerous diseases. Forexample, PDT efficacy can be enhanced in cancerous and non-cancerousconditions (e.g., tuberculosis) using the methods and compositionsdescribed herein (reviewed in, e.g., Agostinis, P. et al. (2011) CACancer J. Clin. 61:250-281).

Anti-Cancer Therapies

In other embodiments, the anti-TCRβV antibody molecule, multispecific ormultifunctional molecule is administered in combination with a low orsmall molecular weight chemotherapeutic agent. Exemplary low or smallmolecular weight chemotherapeutic agents include, but not limited to,13-cis-retinoic acid (isotretinoin, ACCUTANE®), 2-CdA(2-chlorodeoxyadenosine, cladribine, LEUSTATIN™), 5-azacitidine(azacitidine, VIDAZA®), 5-fluorouracil (5-FU, fluorouracil, ADRUCIL®),6-mercaptopurine (6-MP, mercaptopurine, PURINETHOL®), 6-TG(6-thioguanine, thioguanine, THIOGUANINE TABLOID®), abraxane (paclitaxelprotein-bound), actinomycin-D (dactinomycin, COSMEGEN®), alitretinoin(PANRETIN®), all-transretinoic acid (ATRA, tretinoin, VESANOID®),altretamine (hexamethylmelamine, HMM, HEXALEN®), amethopterin(methotrexate, methotrexate sodium, MTX, TREXALL™, RHEUMATREX®),amifostine (ETHYOL®), arabinosylcytosine (Ara-C, cytarabine,CYTOSAR-U®), arsenic trioxide (TRISENOX®), asparaginase (ErwiniaL-asparaginase, L-asparaginase, ELSPAR®, KIDROLASE®), BCNU (carmustine,BiCNU®), bendamustine (TREANDA®), bexarotene (TARGRETIN®), bleomycin(BLENOXANE®), busulfan (BUSULFEX®, MYLERAN®), calcium leucovorin(Citrovorum Factor, folinic acid, leucovorin), camptothecin-11 (CPT-11,irinotecan, CAMPTOSAR®), capecitabine (XELODA®), carboplatin(PARAPLATIN®), carmustine wafer (prolifeprospan 20 with carmustineimplant, GLIADEL® wafer), CCI-779 (temsirolimus, TORISEL®), CCNU(lomustine, CeeNU), CDDP (cisplatin, PLATINOL®, PLATINOL-AQ®),chlorambucil (leukeran), cyclophosphamide (CYTOXAN®, NEOSAR®),dacarbazine (DIC, DTIC, imidazole carboxamide, DTIC-DOME®), daunomycin(daunorubicin, daunorubicin hydrochloride, rubidomycin hydrochloride,CERUBIDINE®), decitabine (DACOGEN®), dexrazoxane (ZINECARD®), DHAD(mitoxantrone, NOVANTRONE®), docetaxel (TAXOTERE®), doxorubicin(ADRIAMYCIN®, RUBEX®), epirubicin (ELLENCE™), estramustine (EMCYT®),etoposide (VP-16, etoposide phosphate, TOPOSAR®, VEPESID®, ETOPOPHOS®),floxuridine (FUDR®), fludarabine (FLUDARA®), fluorouracil (cream)(CARAC™, EFUDEX®, FLUOROPLEX®), gemcitabine (GEMZAR®), hydroxyurea(HYDREA®, DROXIA™, MYLOCEL™), idarubicin (IDAMYCIN®), ifosfamide(IFEX®), ixabepilone (IXEMPRA™), LCR (leurocristine, vincristine, VCR,ONCOVIN®, VINCASAR PFS®), L-PAM (L-sarcolysin, melphalan, phenylalaninemustard, ALKERAN®), mechlorethamine (mechlorethamine hydrochloride,mustine, nitrogen mustard, MUSTARGEN®), mesna (MESNEX™), mitomycin(mitomycin-C, MTC, MUTAMYCIN®), nelarabine (ARRANON®), oxaliplatin(ELOXATIN™), paclitaxel (TAXOL®, ONXAL™), pegaspargase(PEG-L-asparaginase, ONCOSPAR®), PEMETREXED (ALIMTA®), pentostatin(NIPENT®), procarbazine (MATULANE®), streptozocin (ZANOSAR®),temozolomide (TEMODAR®), teniposide (VM-26, VUMON®), TESPA(thiophosphoamide, thiotepa, TSPA, THIOPLEX®), topotecan (HYCAMTIN®),vinblastine (vinblastine sulfate, vincaleukoblastine, VLB, ALKABAN-AQ®,VELBAN®), vinorelbine (vinorelbine tartrate, NAVELBINE®), and vorinostat(ZOLINZA®).

In another embodiment, the anti-TCRβV antibody molecule, multispecificor multifunctional molecule is administered in conjunction with abiologic. Biologics useful in the treatment of cancers are known in theart and a binding molecule of the invention may be administered, forexample, in conjunction with such known biologics. For example, the FDAhas approved the following biologics for the treatment of breast cancer:HERCEPTIN® (trastuzumab, Genentech Inc., South San Francisco, Calif.; ahumanized monoclonal antibody that has anti-tumor activity inHER2-positive breast cancer); FASLODEX® (fulvestrant, AstraZenecaPharmaceuticals, LP, Wilmington, Del.; an estrogen-receptor antagonistused to treat breast cancer); ARIMIDEX® (anastrozole, AstraZenecaPharmaceuticals, LP; a nonsteroidal aromatase inhibitor which blocksaromatase, an enzyme needed to make estrogen); Aromasin® (exemestane,Pfizer Inc., New York, N.Y.; an irreversible, steroidal aromataseinactivator used in the treatment of breast cancer); FEMARA® (letrozole,Novartis Pharmaceuticals, East Hanover, N.J.; a nonsteroidal aromataseinhibitor approved by the FDA to treat breast cancer); and NOLVADEX®(tamoxifen, AstraZeneca Pharmaceuticals, LP; a nonsteroidal antiestrogenapproved by the FDA to treat breast cancer). Other biologics with whichthe binding molecules of the invention may be combined include: AVASTIN®(bevacizumab, Genentech Inc.; the first FDA-approved therapy designed toinhibit angiogenesis); and ZEVALIN® (ibritumomab tiuxetan, Biogen Idec,Cambridge, Mass.; a radiolabeled monoclonal antibody currently approvedfor the treatment of B-cell lymphomas).

In addition, the FDA has approved the following biologics for thetreatment of colorectal cancer: AVASTIN®; ERBITUX® (cetuximab, ImCloneSystems Inc., New York, N.Y., and Bristol-Myers Squibb, New York, N.Y.;is a monoclonal antibody directed against the epidermal growth factorreceptor (EGFR)); GLEEVEC® (imatinib mesylate; a protein kinaseinhibitor); and ERGAMISOL® (levamisole hydrochloride, JanssenPharmaceutica Products, LP, Titusville, N.J.; an immunomodulatorapproved by the FDA in 1990 as an adjuvant treatment in combination with5-fluorouracil after surgical resection in patients with Dukes' Stage Ccolon cancer).

For the treatment of lung cancer, exemplary biologics include TARCEVA®(erlotinib HCL, OSI Pharmaceuticals Inc., Melville, N.Y.; a smallmolecule designed to target the human epidermal growth factor receptor 1(HER1) pathway).

For the treatment of multiple myeloma, exemplary biologics includeVELCADE® Velcade (bortezomib, Millennium Pharmaceuticals, CambridgeMass.; a proteasome inhibitor). Additional biologics include THALIDOMID®(thalidomide, Clegene Corporation, Warren, N.J.; an immunomodulatoryagent and appears to have multiple actions, including the ability toinhibit the growth and survival of myeloma cells and anti-angiogenesis).

Additional exemplary cancer therapeutic antibodies include, but are notlimited to, 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab pegol,alemtuzumab (CAMPATH®, MABCAMPATH®), altumomab pentetate(HYBRI-CEAKER®), anatumomab mafenatox, anrukinzumab (IMA-638),apolizumab, arcitumomab (CEA-SCAN®), bavituximab, bectumomab(LYMPHOSCAN®), belimumab (BENLYSTA®, LYMPHOSTAT-B®), besilesomab(SCINTIMUN®), bevacizumab (AVASTIN®), bivatuzumab mertansine,blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromabpendetide (PROSTASCINT®), catumaxomab (REMOVAB®), CC49, cetuximab (C225,ERBITUX®), citatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan,conatumumab, dacetuzumab, denosumab (PROLIA®), detumomab, ecromeximab,edrecolomab (PANOREX®), elotuzumab, epitumomab cituxetan, epratuzumab,ertumaxomab (REXOMUN®), etaracizumab, farletuzumab, figitumumab,fresolimumab, galiximab, gemtuzumab ozogamicin (MYLOTARG®),girentuximab, glembatumumab vedotin, ibritumomab (ibritumomab tiuxetan,ZEVALIN®), igovomab (INDIMACIS-125®), intetumumab, inotuzumabozogamicin, ipilimumab, iratumumab, labetuzumab (CEA-CIDE®),lexatumumab, lintuzumab, lucatumumab, lumiliximab, mapatumumab,matuzumab, milatuzumab, minretumomab, mitumomab, nacolomab tafenatox,naptumomab estafenatox, necitumumab, nimotuzumab (THERACIM®, THERALOC®),nofetumomab merpentan (VERLUMA®), ofatumumab (ARZERRA®), olaratumab,oportuzumab monatox, oregovomab (OVAREX®), panitumumab (VECTIBIX®),pemtumomab (THERAGYN®), pertuzumab (OMNITARG®), pintumomab, pritumumab,ramucirumab, ranibizumab (LUCENTIS®), rilotumumab, rituximab (MABTHERA®,RITUXAN®), robatumumab, satumomab pendetide, sibrotuzumab, siltuximab,sontuzumab, tacatuzumab tetraxetan (AFP-CIDE®), taplitumomab paptox,tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650,tositumomab (BEXXAR®), trastuzumab (HERCEPTIN®), tremelimumab,tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab(HUMASPECT®), zalutumumab (HUMAX-EGFR®), and zanolimumab (HUMAX-CD4®).

In other embodiments, the anti-TCRβV antibody molecule, multispecific ormultifunctional molecule is administered in combination with a viralcancer therapeutic agent. Exemplary viral cancer therapeutic agentsinclude, but not limited to, vaccinia virus (vvDD-CDSR),carcinoembryonic antigen-expressing measles virus, recombinant vacciniavirus (TK-deletion plus GM-CSF), Seneca Valley virus-001, Newcastlevirus, coxsackie virus A21, GL-ONC1, EBNA1 C-terminal/LMP2 chimericprotein-expressing recombinant modified vaccinia Ankara vaccine,carcinoembryonic antigen-expressing measles virus, G207 oncolytic virus,modified vaccinia virus Ankara vaccine expressing p53, OncoVEX GM-CSFmodified herpes-simplex 1 virus, fowlpox virus vaccine vector,recombinant vaccinia prostate-specific antigen vaccine, humanpapillomavirus 16/18 L1 virus-like particle/AS04 vaccine, MVA-EBNA1/LMP2Inj. vaccine, quadrivalent HPV vaccine, quadrivalent humanpapillomavirus (types 6, 11, 16, 18) recombinant vaccine (GARDASIL®),recombinant fowlpox-CEA(6D)/TRICOM vaccine; recombinantvaccinia-CEA(6D)-TRICOM vaccine, recombinant modified vacciniaAnkara-5T4 vaccine, recombinant fowlpox-TRICOM vaccine, oncolytic herpesvirus NV1020, HPV L1 VLP vaccine V504, human papillomavirus bivalent(types 16 and 18) vaccine (CERVARIX®), herpes simplex virus HF10,Ad5CMV-p53 gene, recombinant vaccinia DF3/MUC1 vaccine, recombinantvaccinia-MUC-1 vaccine, recombinant vaccinia-TRICOM vaccine, ALVACMART-1 vaccine, replication-defective herpes simplex virus type I(HSV-1) vector expressing human Preproenkephalin (NP2), wild-typereovirus, reovirus type 3 Dearing (REOLYSIN®), oncolytic virus HSV1716,recombinant modified vaccinia Ankara (MVA)-based vaccine encodingEpstein-Barr virus target antigens, recombinant fowlpox-prostatespecific antigen vaccine, recombinant vaccinia prostate-specific antigenvaccine, recombinant vaccinia-B7.1 vaccine, rAd-p53 gene,Ad5-delta24RGD, HPV vaccine 580299, JX-594 (thymidine kinase-deletedvaccinia virus plus GM-CSF), HPV-16/18 L1/AS04, fowlpox virus vaccinevector, vaccinia-tyrosinase vaccine, MEDI-517 HPV-16/18 VLP AS04vaccine, adenoviral vector containing the thymidine kinase of herpessimplex virus TK99UN, HspE7, FP253/Fludarabine, ALVAC(2) melanomamulti-antigen therapeutic vaccine, ALVAC-hB7.1, canarypox-hIL-12melanoma vaccine, Ad-REIC/Dkk-3, rAd-IFN SCH 721015, TIL-Ad-INFg,Ad-ISF35, and coxsackievirus A21 (CVA21, CAVATAK®).

In other embodiments, anti-TCRβV antibody molecule, multispecific ormultifunctional molecule is administered in combination with ananopharmaceutical. Exemplary cancer nanopharmaceuticals include, butnot limited to, ABRAXANE® (paclitaxel bound albumin nanoparticles),CRLX101 (CPT conjugated to a linear cyclodextrin-based polymer), CRLX288(conjugating docetaxel to the biodegradable polymer poly(lactic-co-glycolic acid)), cytarabine liposomal (liposomal Ara-C,DEPOCYT™), daunorubicin liposomal (DAUNOXOME®), doxorubicin liposomal(DOXIL®, CAELYX®), encapsulated-daunorubicin citrate liposome(DAUNOXOME®), and PEG anti-VEGF aptamer (MACUGEN®).

In some embodiments, the anti-TCRβV antibody molecule, multispecific ormultifunctional molecule is administered in combination with paclitaxelor a paclitaxel formulation, e.g., TAXOL®, protein-bound paclitaxel(e.g., ABRAXANE®). Exemplary paclitaxel formulations include, but arenot limited to, nanoparticle albumin-bound paclitaxel (ABRAXANE®,marketed by Abraxis Bioscience), docosahexaenoic acid bound-paclitaxel(DHA-paclitaxel, Taxoprexin, marketed by Protarga), polyglutamatebound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX,marketed by Cell Therapeutic), the tumor-activated prodrug (TAP), ANG105(Angiopep-2 bound to three molecules of paclitaxel, marketed byImmunoGen), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizingpeptide EC-1; see Li et al., Biopolymers (2007) 87:225-230), andglucose-conjugated paclitaxel (e.g., 2′-paclitaxel methyl2-glucopyranosyl succinate, see Liu et al., Bioorganic & MedicinalChemistry Letters (2007) 17:617-620).

Exemplary RNAi and antisense RNA agents for treating cancer include, butnot limited to, CALAA-01, siG12D LODER (Local Drug EluteR), andALN-VSP02.

Other cancer therapeutic agents include, but not limited to, cytokines(e.g., aldesleukin (IL-2, Interleukin-2, PROLEUKIN®), alpha Interferon(IFN-alpha, Interferon alfa, INTRON® A (Interferon alfa-2b), ROFERON-A®(Interferon alfa-2a)), Epoetin alfa (PROCRIT®), filgrastim (G-CSF,Granulocyte-Colony Stimulating Factor, NEUPOGEN®), GM-CSF (GranulocyteMacrophage Colony Stimulating Factor, sargramostim, LEUKINE™), IL-11(Interleukin-11, oprelvekin, NEUMEGA®), Interferon alfa-2b (PEGconjugate) (PEG interferon, PEG-INTRON™), and pegfilgrastim(NEULASTA™)), hormone therapy agents (e.g., aminoglutethimide(CYTADREN®), anastrozole (ARIIVIIDEX®), bicalutamide (CASODEX®),exemestane (AROMASIN®), fluoxymesterone (HALOTESTIN®), flutamide(EULEXIN®), fulvestrant (FASLODEX®), goserelin (ZOLADEX®), letrozole(FEMARA®), leuprolide (ELIGARD™, LUPRON®, LUPRON DEPOT®, VIADUR™),megestrol (megestrol acetate, MEGACE®), nilutamide (ANANDRON®,NILANDRON®), octreotide (octreotide acetate, SANDOSTATIN®, SANDOSTATINLAR®), raloxifene (EVISTA®), romiplostim (NPLATE®), tamoxifen(NOVALDEX®), and toremifene (FARESTON®)), phospholipase A2 inhibitors(e.g., anagrelide (AGRYLIN®)), biologic response modifiers (e.g., BCG(THERACYS®, TICE®), and Darbepoetin alfa (ARANESP®)), target therapyagents (e.g., bortezomib (VELCADE®), dasatinib (SPRYCEL™), denileukindiftitox (ONTAK®), erlotinib (TARCEVA®), everolimus (AFINITOR®),gefitinib (IRESSA®), imatinib mesylate (STI-571, GLEEVEC™), lapatinib(TYKERB®), sorafenib (NEXAVAR®), and SU11248 (sunitinib, SUTENT®)),immunomodulatory and antiangiogenic agents (e.g., CC-5013 (lenalidomide,REVLIMID®), and thalidomide (THALOMID®)), glucocorticosteroids (e.g.,cortisone (hydrocortisone, hydrocortisone sodium phosphate,hydrocortisone sodium succinate, ALA-CORT®, HYDROCORT ACETATE®,hydrocortone phosphate LANACORT®, SOLU-CORTEF®), decadron(dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate,DEXASONE®, DIODEX®, HEXADROL®, MAXIDEX®), methylprednisolone(6-methylprednisolone, methylprednisolone acetate, methylprednisolonesodium succinate, DURALONE®, MEDRALONE®, MEDROL®, M-PREDNISOL®,SOLU-MEDROL®), prednisolone (DELTA-CORTEF®, ORAPRED®, PEDIAPRED®,PRELONE®), and prednisone (DELTASONE®, LIQUID PRED®, METICORTEN®,ORASONE®)), and bisphosphonates (e.g., pamidronate (AREDIA®), andzoledronic acid (ZOMETA®))

In some embodiments, the anti-TCRβV antibody molecule, multispecific ormultifunctional molecule is used in combination with a tyrosine kinaseinhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor). Exemplarytyrosine kinase inhibitor include, but are not limited to, an epidermalgrowth factor (EGF) pathway inhibitor (e.g., an epidermal growth factorreceptor (EGFR) inhibitor), a vascular endothelial growth factor (VEGF)pathway inhibitor (e.g., an antibody against VEGF, a VEGF trap, avascular endothelial growth factor receptor (VEGFR) inhibitor (e.g., aVEGFR-1 inhibitor, a VEGFR-2 inhibitor, a VEGFR-3 inhibitor)), aplatelet derived growth factor (PDGF) pathway inhibitor (e.g., aplatelet derived growth factor receptor (PDGFR) inhibitor (e.g., aPDGFR-ß inhibitor)), a RAF-1 inhibitor, a KIT inhibitor and a RETinhibitor. In some embodiments, the anti-cancer agent used incombination with the AHCM agent is selected from the group consistingof: axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™,AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®),gefitinib ORES SA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib(TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272),nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®,SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474),vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab(AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab(VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib(NEXAVAR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®),ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BMW2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607,ABT-869, MP470, BIM 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265,DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121,XL-184, XL-647, XL228, AEE788, AG-490, AST-6, BMS-599626, CUDC-101,PD153035, pelitinib (EKB-569), vandetanib (zactima), WZ3146, WZ4002,WZ8040, ABT-869 (linifanib), AEE788, AP24534 (ponatinib), AV-951(tivozanib), axitinib, BAY 73-4506 (regorafenib), brivanib alaninate(BMS-582664), brivanib (BMS-540215), cediranib (AZD2171), CHIR-258(dovitinib), CP 673451, CYC116, E7080, Ki8751, masitinib (AB1010),MGCD-265, motesanib diphosphate (AMG-706), MP-470, OSI-930, PazopanibHydrochloride, PD173074, Sorafenib Tosylate (Bay 43-9006), SU 5402,TSU-68 (SU6668), vatalanib, XL880 (GSK1363089, EXEL-2880). Selectedtyrosine kinase inhibitors are chosen from sunitinib, erlotinib,gefitinib, or sorafenib. In one embodiment, the tyrosine kinaseinhibitor is sunitinib.

In one embodiment, the anti-TCRβV antibody molecule, multispecific ormultifunctional molecule is administered in combination with one of moreof: an anti-angiogenic agent, or a vascular targeting agent or avascular disrupting agent. Exemplary anti-angiogenic agents include, butare not limited to, VEGF inhibitors (e.g., anti-VEGF antibodies (e.g.,bevacizumab); VEGF receptor inhibitors (e.g., itraconazole); inhibitorsof cell proliferatin and/or migration of endothelial cells (e.g.,carboxyamidotriazole, TNP-470); inhibitors of angiogenesis stimulators(e.g., suramin), among others. A vascular-targeting agent (VTA) orvascular disrupting agent (VDA) is designed to damage the vasculature(blood vessels) of cancer tumors causing central necrosis (reviewed in,e.g., Thorpe, P. E. (2004) Clin. Cancer Res. Vol. 10:415-427). VTAs canbe small-molecule. Exemplary small-molecule VTAs include, but are notlimited to, microtubule destabilizing drugs (e.g., combretastatin A-4disodium phosphate (CA4P), ZD6126, AVE8062, Oxi 4503); and vadimezan(ASA404).

Immune Checkpoint Inhibitors

In other embodiments, methods described herein comprise use of an immunecheckpoint inhibitor in combination with the anti-TCRβV antibodymolecule, multispecific or multifunctional molecule. The methods can beused in a therapeutic protocol in vivo.

In embodiments, an immune checkpoint inhibitor inhibits a checkpointmolecule. Exemplary checkpoint molecules include but are not limited toCTLA4, PD1, PD-L1, PD-L2, TIM3, LAG3, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), BTLA, KIR, MHC classI, MHC class II, GALS, VISTA, BTLA, TIGIT, LAIR1, and A2aR. See, e.g.,Pardoll. Nat. Rev. Cancer 12.4 (2012):252-64, incorporated herein byreference.

In embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor,e.g., an anti-PD-1 antibody such as Nivolumab, Pembrolizumab orPidilizumab. Nivolumab (also called MDX-1106, MDX-1106-04, ONO-4538, orBMS-936558) is a fully human IgG4 monoclonal antibody that specificallyinhibits PD1. See, e.g., U.S. Pat. No. 8,008,449 and WO2006/121168.Pembrolizumab (also called Lambrolizumab, MK-3475, MK03475, SCH-900475or KEYTRUDA®; Merck) is a humanized IgG4 monoclonal antibody that bindsto PD-1. See, e.g., Hamid, O. et al. (2013) New England Journal ofMedicine 369 (2): 134-44, U.S. Pat. No. 8,354,509 and WO2009/114335.Pidilizumab (also called CT-011 or Cure Tech) is a humanized IgG1kmonoclonal antibody that binds to PD1. See, e.g., WO2009/101611. In oneembodiment, the inhibitor of PD-1 is an antibody molecule having asequence substantially identical or similar thereto, e.g., a sequence atleast 85%, 90%, 95% identical or higher to the sequence of Nivolumab,Pembrolizumab or Pidilizumab. Additional anti-PD1 antibodies, e.g., AMP514 (Amplimmune), are described, e.g., in U.S. Pat. No. 8,609,089, US2010028330, and/or US 20120114649.

In some embodiments, the PD-1 inhibitor is an immunoadhesin, e.g., animmunoadhesin comprising an extracellular/PD-1 binding portion of a PD-1ligand (e.g., PD-L1 or PD-L2) that is fused to a constant region (e.g.,an Fc region of an immunoglobulin). In embodiments, the PD-1 inhibitoris AMP-224 (B7-DCIg, e.g., described in WO2011/066342 andWO2010/027827), a PD-L2 Fc fusion soluble receptor that blocks theinteraction between B7-H1 and PD-1.

In embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor,e.g., an antibody molecule. In some embodiments, the PD-L1 inhibitor isYW243.55.570, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105. In someembodiments, the anti-PD-L1 antibody is MSB0010718C (also calledA09-246-2; Merck Serono), which is a monoclonal antibody that binds toPD-L1. Exemplary humanized anti-PD-L1 antibodies are described, e.g., inWO2013/079174. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1antibody, e.g., YW243.55.S70. The YW243.55.S70 antibody is described,e.g., in WO 2010/077634. In one embodiment, the PD-L1 inhibitor isMDX-1105 (also called BMS-936559), which is described, e.g., inWO2007/005874. In one embodiment, the PD-L1 inhibitor is MDPL3280A(Genentech/Roche), which is a human Fc-optimized IgG1 monoclonalantibody against PD-L1. See, e.g., U.S. Pat. No. 7,943,743 and U.SPublication No.: 20120039906. In one embodiment, the inhibitor of PD-L1is an antibody molecule having a sequence substantially identical orsimilar thereto, e.g., a sequence at least 85%, 90%, 95% identical orhigher to the sequence of YW243.55.S70, MPDL3280A, MEDI-4736,MSB-0010718C, or MDX-1105.

In embodiments, the immune checkpoint inhibitor is a PD-L2 inhibitor,e.g., AMP-224 (which is a PD-L2 Fc fusion soluble receptor that blocksthe interaction between PD1 and B7-H1. See, e.g., WO2010/027827 andWO2011/066342.

In one embodiment, the immune checkpoint inhibitor is a LAG-3 inhibitor,e.g., an anti LAG-3 antibody molecule. In embodiments, the anti-LAG-3antibody is BMS-986016 (also called BMS986016; Bristol-Myers Squibb).BMS-986016 and other humanized anti-LAG-3 antibodies are described,e.g., in US 2011/0150892, WO2010/019570, and WO2014/008218.

In embodiments, the immune checkpoint inhibitor is a TIM-3 inhibitor,e.g., anti-TIM3 antibody molecule, e.g., described in U.S. Pat. No.8,552,156, WO 2011/155607, EP 2581113 and U.S Publication No.:2014/044728.

In embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor,e.g., anti-CTLA-4 antibody molecule. Exemplary anti-CTLA4 antibodiesinclude Tremelimumab (IgG2 monoclonal antibody from Pfizer, formerlyknown as ticilimumab, CP-675,206); and Ipilimumab (also called MDX-010,CAS No. 477202-00-9). Other exemplary anti-CTLA-4 antibodies aredescribed, e.g., in U.S. Pat. No. 5,811,097.

CRS Grading

In some embodiments, CRS can be graded in severity from 1-5 as follows.Grades 1-3 are less than severe CRS. Grades 4-5 are severe CRS. ForGrade 1 CRS, only symptomatic treatment is needed (e.g., nausea, fever,fatigue, myalgias, malaise, headache) and symptoms are not lifethreatening. For Grade 2 CRS, the symptoms require moderate interventionand generally respond to moderate intervention. Subjects having Grade 2CRS develop hypotension that is responsive to either fluids or onelow-dose vasopressor; or they develop grade 2 organ toxicity or mildrespiratory symptoms that are responsive to low flow oxygen (<40%oxygen). In Grade 3 CRS subjects, hypotension generally cannot bereversed by fluid therapy or one low-dose vasopressor. These subjectsgenerally require more than low flow oxygen and have grade 3 organtoxicity (e.g., renal or cardiac dysfunction or coagulopathy) and/orgrade 4 transaminitis. Grade 3 CRS subjects require more aggressiveintervention, e.g., oxygen of 40% or higher, high dose vasopressor(s),and/or multiple vasopressors. Grade 4 CRS subjects suffer fromimmediately life-threatening symptoms, including grade 4 organ toxicityor a need for mechanical ventilation. Grade 4 CRS subjects generally donot have transaminitis. In Grade 5 CRS subjects, the toxicity causesdeath. Sets of criteria for grading CRS are provided herein as Table 5,Table 6, and Table 7. Unless otherwise specified, CRS as used hereinrefers to CRS according to the criteria of Table 6.

In embodiments, CRS is graded according to Table 5:

TABLE 5 CRS grading Gr1 Supportive care only Gr2 IV therapies +/−hospitalization. Gr3 Hypotension requiring IV fluids or low-dosevasoactives or hypoxemia requiring oxygen, CPAP, or BIPAP. Gr4Hypotension requiring high-dose vasoactives or hypoxemia requiringmechanical ventilation. Gr 5 Death

TABLE 6 CTCAE v 4.0 CRS grading scale CRS grade Characteristics Grade 1Mild; No infusion interruption; No intervention Grade 2 Infusioninterruption indicated but responds promptly to symptomatic treatment(e.g., antihistamines, NSAIDS, narcotics, IV fluids); prophylacticmedications indicated for <=24 hrs Grade 3 Prolonged (e.g., not rapidlyresponsive to symptomatic medications and/or brief interruption ofinfusion); recurrence of symptoms following initial improvement;hospitalization indicated for clinical sequelae (e.g., renal impairment,pulmonary infiltrates) Grade 4 Life threatening consequences; pressor orventilator support

TABLE 7 NCI CRS grading scale CRS grade Characteristics Grade 1 Symptomsare not life threatening and require symptomatic treatment only; e.g.,fever, nausea, fatigue, headache, myalgias, malaise Grade 2 Symptomsrequire and respond to moderate intervention; Oxygen requirement <40% orhypotension responsive to fluids or low dose pressors or Grade 2 organtoxicity Grade 3 Symptoms require and respond to aggressiveintervention; Oxygen requirement >=40% or Hypotension requiring highdose or multiple pressors or grade 3 organ toxicity or grade 4transaminitis Grade 4 Life threatening symptoms Requirement forventilator support or Grade 4; organ toxicity (excluding transaminitis)

EXAMPLES Example 1. Humanization of α-TRBV6-5 Antibody Clone Antibody A

The germline for the mouse α-TCRβ antibody clone Antibody A VH and VLwere assigned using IMGT nomenclature, with CDR regions defined by acombined Kabat and Chothia classification. SEQ ID NO: 1 and SEQ ID NO: 2are the Antibody A VH and VL sequences respectively where the VHgermline is mouse IGHV1S12*01 and the VL germline is mouse IGKV6-15*01.SEQ ID NOs: 3-5 are the Antibody A VH CDR regions 1-3 respectively andSEQ ID NOs: 6-8 correspond to the VL CDR regions 1-3 (as described inTable 1).

Humanization of the Antibody A VH and VL sequences was done separatelyusing similar methodology. Amino acids positions were identified in theframework regions which were important for the success of CDR grafting.Human germline sequences were identified which preserved the necessaryresidues and contained a high amount of overall identity. When the humangermline framework sequence did not contain a matching important aminoacid, it was back mutated to match the mouse sequence. CDR regions weregrafted onto the human germline unchanged. The Antibody A VH washumanized into human IGHV1-69*01 and the Antibody A VL was humanizedinto IGKV1-17*01 and IGKV1-27*01. All 3 humanized sequences wereconfirmed to contain no introduced potential negative post translationalmodification sites such as NG, DG, NS, NN, DS, NT, NXS, or NXT as aresult of the humanization process. SEQ ID NO: 9 is the humanizedAntibody A-H.1 VH and SEQ ID NOs: 10 and 11 are the humanized VLIGKV1-17*01 and IGKV1-27*01 germlines respectively (as described inTable 1). FIGS. 1A and 1B show the murine and humanized sequences withannotations depicting the CDR and framework regions (FR).

Example 2: Humanization of α-TRBV12-3 and TRBV12-4 Antibody CloneAntibody B

The germline for the mouse α-TCRβ antibody clone Antibody B VH and VLwere assigned using IMGT nomenclature, with CDR regions defined by acombined Kabat and Chothia classification. SEQ ID NO: 15 and SEQ ID NO:16 are the Antibody B VH and VL sequences respectively where the VHgermline is mouse IGHV5-17*02 and the VL germline is mouse IGKV4-50*01.SEQ ID NOs: 17-19 are the B-H VH CDR regions 1-3 respectively and SEQ IDNOs: 20-22 are the B-H VL CDR regions 1-3 (as described in Table 2).

The method applied to humanize Antibody A described in Example 1 wasused to humanize Antibody B. The Antibody B VH was humanized into humanIGHV3-30*01, IGHV3-48*01, and IGHV3-66*01 and the Antibody B VL washumanized into human IGKV1-9*01, IGKV1-39*01, IGKV3-15*01, IGLV1-47*01and IGLV3-10*01. SEQ ID NOs: 23-25 are the B-H.1A, B-H.1B, and B-H.1Chumanized heavy chains and SEQ ID NOs: 26-30 are the B-H.1D, B-H.1E,B-H.1F, B-H.1G and B-H.1H humanized light chains (as described in Table2). FIGS. 2A and 2B show the murine and humanized sequences withannotations depicting the CDR and framework regions (FR).

Example 3: Characteristics of Anti-TCRβV Antibodies

Introduction

Current bispecific constructs designed to redirect T cells to promotetumor cell lysis for cancer immunotherapy typically utilize single chainvariable fragments (scFVs) that are derived from monoclonal antibodies(mAb) directed against the CD3e subunit of the T cell receptor (TCR).However, there are limitations to this approach which may prevent thefull realization of the therapeutic potential for such bispecificconstructs. Previous studies have shown that, e.g., low “activating”doses of anti-CD3e mAb can cause long-term T cell dysfunction and exertimmunosuppressive effects. In addition, anti-CD3e mAbs bind to all Tcells and thus activate equally all T cells, which has been associatedwith the first dose side effects of anti-CD3e mAbs that result frommassive T cell activation. These large number of activated T cellssecrete substantial amounts of cytokines, the most important of which isInterferon gamma (IFNg). This excess amount of IFNg in turn, e.g.,activates macrophages which then can overproduce proinflammatorycytokines such as IL-1, IL-6 and TNF-alpha, causing a “cytokine storm”known as the cytokine release syndrome (CRS). Thus, it might beadvantageous to develop antibodies that are capable of binding andactivating only a subset of necessary effector T cells to reduce theCRS.

Results

To that end, antibodies directed to the variable chain of the betasubunit of TCR (TCR Vb) were identified. These anti-TCR Vb antibodiesbind and activate a subset of T cells, but with, e.g., no or markedlyreduced CRS. Using plate-bound anti-TCR Vb13.1 mAbs (A-H.1 and A-H.2) itwas shown that a population of T cells, defined by positive stainingwith A-H.1, can be expanded (from ˜5% of T cells on day 0 to almost 60%of total T cells on day 6 of cell culture) (FIGS. 4A-4C). For thisexperiment, human CD3+ T cells were isolated using magnetic-beadseparation (negative selection) and activated with immobilized(plate-coated) A-H.1 or OKT3 (anti-CD3e) antibodies at 100 nM for 6days. The expanded Vb13.1+ T cells display cytolytic activity againsttransformed cell line RPMI-8226 when co-cultured with purified CD3+ Tcells (FIGS. 5A-5B).

Next, the ability of PBMCs activated by anti-TCR VB antibodies toproduce cytokines was assessed. The cytokine production of PBMCsactivated with anti-TCR VB antibodies was compared to the cytokineproduction of PBMCs activated with: (i) anti-CD3e antibodies (OKT3 orSP34-2); (ii) anti-TCR V alpha (TCR VA) antibodies including anti-TCR VA12.1 antibody 6D6.6, anti-TCR VA24JA18 antibody 6B11; (iii) anti-TCRalpha beta antibody T10B9; and/or (iv) isotype control (BGM0109). Theanti-TCR VB antibodies tested include: humanized anti-TCRVB 13.1antibodies (A-H.1, or A-H.2), murine anti-TCR VB5 antibody Antibody E,murine anti-TCR VB8.1 antibody Antibody B, and murine anti-TCR VB12antibody Antibody D. BGM0109 comprises the amino acid sequence of

(SEQ ID NO: 3282) METDTLLLWVLLLWVPGSTGGLNDIFEAQKIEWHEGGGGSEPRTDTDTCPNPPDPCPTCPTPDLLGGPSVFIFPPKPKDVLMISLTPKITCVVVDVSEEEPDVQFNWYVNNVEDKTAQTETRQRQYNSTYRVVSVLPIKHQDWMSGKVFKCKVNNNALPSPIEKTISKPRGQVRVPQIYTFPPPIEQTVKKDVSVTCLVTGFLPQDIHVEWESNGQPQPEQNYKNTQPVLDSDGSYFLYSKLNVPKSRWDQGDSFTCSVIHEALHNHHMTKTISRSLGNGGG GS.

As shown in FIG. 6A, when plate-bound A-H.1 or A-H.2, or anti-CD3eantibodies (OKT3 or SP34-2) were used to activate human PBMCs, the Tcell cytokine IFNg was induced (FIG. 6A). All anti-TCR VB antibodiestested had a similar effect on the production of IFNg (FIG. 6B). Theanti-TCR VA antibodies did not induce similar IFNg production.

With respect to IL-2 production, PBMCs activated with A-H.1 and A-H.2resulted in increased IL-2 production (FIG. 7A) with delayed kinetics(FIG. 7B) as compared to PBMCs activated with anti-CD3e antibodies (OKT3or SP34-2). FIG. 7B shows that anti-TCR VB antibody activated PBMCsdemonstrate peak production of IL-2 at Day 5 or Day 6 post-activation(incubation with plate-coated antibodies). In contrast, IL-2 productionin PBMCs activated with OKT3 peaked at day 2 post-activation. As withIFNG, the IL-2 effect (e.g., enhanced production of IL-2 and delayedkinetics) was similar across all anti-TCR VB antibodies tested (FIG.7B).

The production of cytokines IL-6, IL-1β and TNF-alpha which areassociated with “cytokine storms” (and accordingly CRS) was alsoassessed under similar conditions. FIGS. 8A, 9A and 10A shows that whilePBMCs activated with anti-CD3e antibodies demonstrate production of IL-6(FIG. 8A), TNF-alpha (FIG. 9A) and IL-1β (FIG. 10A), no or littleinduction of these cytokines was observed with PBMCs activated withA-H.1 or A-H.2. As shown in FIGS. 9B and 10B, TNF-alpha and IL-1βproduction was not induced by activation of PBMCs with any of theanti-TCR VB antibodies.

It was further noted that the kinetics of IFNg production byA-H.1-activated CD3+ T cells was delayed relative to those produced byCD3+ T cells activated by anti-CD3e mAbs (OKT3 and SP34-2) (FIGS. 11Aand 11B).

Finally, it was observed that the subset of memory effector T cellsknown as T_(EMRA) was preferentially expanded in CD8+ T cells activatedby A-H.1 or A-H.2 (FIG. 12). Isolated human PBMCs were activated withimmobilized (plate-coated) anti-CD3e or anti-TCR Vβ13.1 at 100 nM for6-days. After a 6-day incubation, T-cell subsets were identified by FACSstaining for surface markers for Naive T cell (CD8+, CD95−, CD45RA+,CCR7+), T stem cell memory (TSCM; CD8+, CD95+, CD45RA+, CCR7+), Tcentral memory (Tcm; CD8+, CD95+, CD45RA−, CCR7+), T effector memory(Tem; CD8+, CD95+, CD45RA−, CCR7−), and T effector memory re-expressingCD45RA (Temra; CD8+, CD95+, CD45RA+, CCR7−). Human PBMCs activated byanti-TCR Vβ13.1 antibodies (A-H.1 or A-H.2) increased CD8+ TSCM andTemra T cell subsets when compared to PBMCs activated by anti-CD3eantibodies (OKT3 or SP34-2). Similar expansion was observed with CD4+ Tcells.

Conclusion

The data provided in this Example show that antibodies directed againstTCR Vb can, e.g., preferentially activate a subset of T cells, leadingto an expansion of T_(EMRA), which can, e.g., promote tumor cell lysisbut not CRS. Thus, bispecific constructs utilizing either a Fab or scFVor a peptide directed to the TCR Vb can, e.g., be used to activate andredirect T cells to promote tumor cell lysis for cancer immunotherapy,without, e.g., the harmful side-effects of CRS associated with anti-CD3etargeting.

Example 4: On-Target T Cell Mediated Cytotoxicity of Multiple Myeloma(MM) Cells with a Dual-Targeting Antibody Molecule Against BCMA and a TCell Engager

This example shows on-target T cell mediated cytotoxicity of multiplemyeloma (MAUI) cells with dual-targeting antibody molecules thatrecognize a T cell engager, e.g., TCRVb, on T cells and BCMA on MMcells.

As shown in FIG. 13A, purified human T cells activated with plate-boundanti-TCRVb antibody for 5 days proliferate at a higher rate thanpurified human T cells activated with plate-bound anti-CD3 (OKT3)antibody. Anti-TCRVb antibody stimulation of T cells resulted inselective expansion of CD45RA+ effector memory CD8+ and CD4+ T cells(TEMRA) cells (FIG. 13B). Both CD8+ and CD4+ Temra cell populationsexpanded more when stimulated with an anti-TCRVb antibody, compared tounstimulated cells or cells stimulated with an anti-CD3 (SP34) antibody.Anti-TCRVb antibodies resulted in delayed secretion of IFN-g by PBMCsstimulated with an anti-TCRVb antibody compared to PBMCs stimulated withanti-CD3 antibodies (FIG. 13C). Additionally, T cells stimulated withanti-TCRVb antibody or anti-CD3 antibodies resulted in comparable lysisof multiple myeloma target cells, as shown in FIG. 13D. As shown inFIGS. 13E-13F, T cells stimulated for 5 days with 100 ng/ml plate-boundan anti-TCRVb antibody, or an anti-CD3 antibody secreted perforin andGranzyme B.

Activation of PBMCs with anti-TCRVb antibody resulted in higherproduction and/or secretion of IL-2 and/or IL-15 compared to PBMCsactivated with an anti-OKT3 antibody (FIG. 14A). Anti-TCRVb antibodyactivated of PBMCs also resulted in expansion and/or survival, e.g.,proliferation of Natural Killer (NK) cells (FIG. 14B). In comparison,PBMCS activated with an anti-OKT3 antibody did not result in NK cellexpansion. Further, as described in Example 3, PBMCs activated with ananti-TCRVb antibody did not result in the production of cytokines IL-6,IL-1β and TNF-alpha which are associated with CRS (FIG. 15). These invitro characterization studies show that in some embodiments, anti-TCRVbantibodies, e.g., activate and/or stimulate, T cells to promote T cellkilling as evidenced by target cell lysis, perforin secretion andgranzyme B secretion, and secretion of IFN-g with, e.g., delayedkinetics.

Next, the ability of a dual-targeting antibody molecule (Molecule I),which targets BCMA on one arm and TCRVb on the other arm, to target andkill multiple myeloma (MM) cells was tested. Healthy donor PBMCs wereco-incubated with the RMPI8226 MM cell line and one of the followingdual-targeting antibody molecules: BCMA-TCRVb (Molecule I), BCMA-CD3, orControl-TCRVb; or an isotype control Target cell lysis was then assessedusing flow cytometry. As shown in FIG. 16A, the dual-targetingBCMA-TCRVb antibody molecule (Molecule I) resulted in killing of MMcells in vitro.

The dual-targeting BCMA-TCRVb antibody molecule (Molecule I) was furthertested in vivo for its ability to inhibit MM tumor growth in a MM mousemodel. The NCI-H929 cell line was injected in NOD-scid IL2rγnull (NSG)recipient mice on Day 0 followed by delivery of PBMCs on Day 9. On Days12, 15, 18 and 21, the dual-targeting BCMA-TCRVb antibody molecule(Molecule I) was administered via intraperitoneal injection at a dose of0.5 mg/kg. FIG. 16B shows prevention, e.g., inhibition, of MM tumorgrowth in vivo with the dual-targeting BCMA-TCRVb antibody molecule(Molecule I). These results demonstrate that in some embodiments thedual-targeting BCMA-TCRVb antibody molecule, e.g., can kill tumor cells,e.g., MM tumor cells, in vitro and in vivo. Accordingly, in someembodiments, a dual-targeting BCMA-TCRVb antibody molecule can be used,e.g., as a therapy for cancer, e.g., a hematological cancer, e.g., MM.

Example 5: In Vitro Cytotoxicity of a Dual-Targeting Antibody MoleculeAgainst FcRH5 and a T Cell Engager

This example shows in vitro cytotoxicity on multiple myeloma (MM) cellswith a dual-targeting antibody molecule that recognizes a T cellengager, e.g., TCRVb, on T cells and FcRH5 on MM cells. Healthy donorPBMCs or purified T cells were co-incubated with the MOL8M MM cell lineand a dual-targeting antibody molecule which targets FcRH5 on one armand TCRVb on the other arm (Molecule E), or with an isotype controlantibody. Target cell lysis was then assessed using flow cytometry. Asshown in FIG. 17, the dual targeting FcRH5-TCRVb molecule (Molecule E)resulted in killing of MM cells by both purified T cells or PBMCs. Thisshows that the dual targeting FcRH5-TCRVb molecule can target andpromote killing of MM cells by immune cells, e.g., in PBMCs, including Tcells.

Example 6: Characteristics of Anti-TCR Vβ8a Antibodies

This Example shows in vitro characterization of anti-TCR Vβ8a antibodies(B-H.1). TCR Vβ8 is also referred to as TCR Vβ12 (as described in Table8). Isolated human PBMCs were activated with immobilized (plate-coated)anti-CD3ϵ or anti-TCR Vβ8a at 100 nM, and cell culture supernatants werecollected on day 1, 2, 3, 5, 6 and 8 post stimulation. Cytokines (IFNγ,IL-2, TNFα, IL-1β or IL-6) were measured using MSD technology platform(MesoScale Discovery) as described in the manufacturer's protocol.

As shown in FIGS. 18A-18B, Human PBMCs activated by anti-TCR Vβ8aantibodies (B-H.1) produce similar or reduced levels of IFNγ (FIG. 18A)and higher levels IL-2 (FIG. 18B) when compared to those activated byanti-CD3ϵ antibodies (OKT3 or SP34-2).

FIGS. 19A-19B show that human PBMCs activated by anti-TCR Vβ8aantibodies (B-H.1) do not produce significant levels of IL-6, or IL1b.Activation of human PBMCs with anti-TCR Vβ8a antibodies (B-H.1) alsoresults in lesser TNFa when compared to PBMCs activated by anti-CD3ϵantibodies (OKT3 or SP34-2) (see FIG. 19C).

In summary, as shown in Example 3, this Example shows that anti-TCR Vβ8aantibodies can, e.g., preferentially induce expression of T cellcytokines, e.g., IL-2 and IFNg, but not production of cytokines IL-6,IL-1β and TNF-alpha which are associated with “cytokine storms” (andaccordingly CRS).

Example 7: Characteristics of Anti-TCRβV Antibody D Antibody

This Example describes characterization of anti-TCRβV antibodies whichcan bind and activate a subset of T cells, but with, e.g., no ormarkedly reduced, CRS.

Human PBMCs were isolated from whole blood followed by solid-phase(plate-coated) stimulation with anti-TCR Vβ12 antibody (Antibody D) oranti-CD3e antibodies (OKT3) at 100 nM. Supernatant was collected on Days1, 2, 3, 5, or 6 followed by multiplex cytokine analysis for IFNg, IL-2,IL-6, IL-1beta, or TNFalpha. The data was quantified using MSD (MesoScale Discovery) platform, following the manufacturer's protocol.

As shown in FIG. 20A, when plate-bound anti-TCR Vβ12 antibody (AntibodyD) or anti-CD3e antibodies (OKT3) were used to activate human PBMCs, theT cell cytokine IFNg was induced. With respect to IL-2 production, PBMCsactivated with anti-TCR Vβ12 antibody (Antibody D) resulted in increasedIL-2 production with delayed kinetics (FIG. 20B) as compared to PBMCsactivated with anti-CD3e antibodies (OKT3).

The production of cytokines IL-6, IL-1β and TNF-alpha which areassociated with “cytokine storms” (and accordingly CRS) was alsoassessed under similar conditions. FIGS. 20C-20E show that that whilePBMCs activated with anti-CD3e antibodies demonstrate production of IL-6(FIG. 20D), TNF-alpha (FIG. 20C) and IL-1β (FIG. 20E), no or littleinduction of these cytokines was observed with PBMCs activated withanti-TCR Vβ12 antibody (Antibody D).

The data provided in this Example show that antibodies directed againstTCR Vβ can, e.g., preferentially activate a subset of T cells, and donot results in induction of cytokines associated with cytokine storms orCRS.

Example 8: Characteristics of Anti-TCRβV Antibody E

This Example describes characterization of anti-TCRβV antibodies whichcan bind and activate a subset of T cells, but with, e.g., no ormarkedly reduced, CRS.

Human PBMCs were isolated from whole blood followed by solid-phase(plate-coated) stimulation with anti-TCR Vβ5 antibody (Antibody E) oranti-CD3e antibodies (OKT3 and SP34-2), each at 100 nM. Supernatant wascollected on Days 1, 3, 5, or 7 followed by multiplex cytokine analysisfor IFNg, IL-2, IL-6, IL-1beta, IL-10 or TNFalpha. The data wasquantified using MSD (Meso Scale Discovery) platform, following themanufacturer's protocol.

As shown in FIG. 21A, when plate-bound anti-TCR Vβ5 antibody (AntibodyE) or anti-CD3e antibodies (OKT3 and SP34-2) were used to activate humanPBMCs, the T cell cytokine IFNg was induced. With respect to IL-2production, PBMCs activated with anti-TCR Vβ5 antibody (Antibody E)resulted in increased IL-2 production with delayed kinetics (FIG. 21B)as compared to PBMCs activated with anti-CD3e antibodies (OKT3 orSP34-2).

The production of cytokines IL-6, IL-13, IL-10 and TNF-alpha which areassociated with “cytokine storms” (and accordingly CRS) was alsoassessed under similar conditions. FIGS. 22A-22D show that that whilePBMCs activated with anti-CD3e antibodies demonstrate production ofIL-1β, (FIG. 22A), IL-6 (FIG. 22B), TNF-alpha (FIG. 22C) and IL-10 (FIG.22D), no or little induction of these cytokines was observed with PBMCsactivated with anti-TCR Vβ5 antibody (Antibody E).

The data provided in this Example show that antibodies directed againstTCR Vβ can, e.g., preferentially activate a subset of T cells, and donot results in induction of cytokines associated with cytokine storms orCRS.

Example 9: Characteristics of a Dual-Targeting Antibody Molecule AgainstBCMA and TCRβV

This Example describes characterization of a dual targeting antibody(e.g., a bispecific molecule) comprising an anti-TCRβV binding moietyand a BCMA binding moiety (Molecule H) which can bind and activate asubset of T cells, but with, e.g., no or markedly reduced, CRS.

Human PBMCs were isolated from whole blood followed by solid-phase(plate-coated) stimulation with an anti-TCRβV×BCMA bispecific molecule(Molecule H) or anti-CD3e antibodies (OKT3), each at 100 nM. Supernatantwas collected on Days 1, 2, 3, or 5 followed by multiplex cytokineanalysis for IFNg, IL-2, IL-6, IL-1beta, IL-10 or TNFalpha. The data wasquantified using MSD (Meso Scale Discovery) platform, following themanufacturer's protocol.

As shown in FIG. 23A, when plate-bound anti-TCRβV×BCMA bispecificmolecule (Molecule H) or anti-CD3e antibodies (OKT3) were used toactivate human PBMCs, the T cell cytokine IFNg was induced. With respectto IL-2 production, PBMCs activated with anti-TCRβV×BCMA bispecificmolecule (Molecule H) resulted in increased IL-2 production (FIG. 23B)as compared to PBMCs activated with anti-CD3e antibodies (OKT3).

The production of cytokines IL-6, IL-1β, IL-10 and TNF-alpha which areassociated with “cytokine storms” (and accordingly CRS) was alsoassessed under similar conditions. FIGS. 23C-E show that that whilePBMCs activated with anti-CD3e antibodies demonstrate production ofIL-1β (FIG. 23C), IL-6 (FIG. 23D), TNF-alpha (FIG. 23D) and IL-10 (FIG.23E), no or little induction of these cytokines was observed with PBMCsactivated with anti-TCRβV×BCMA bispecific molecule (Molecule H).

The data provided in this Example show that antibodies directed againstTCR Vβ can, e.g., preferentially activate a subset of T cells, and donot result in induction of cytokines associated with cytokine storm orCRS.

Example 10: Cytokine and Chemokine Profile of Anti-TCRVb Antibodies

This Examples describes cytokines and chemokines secreted by PBMCsfollowing activation by anti-TCR Vβ antibodies.

Human PBMCs were isolated from whole blood followed by solid-phase(plate-coated) stimulation with an anti-TCRβV antibodies (A-H.1, B-H.1),or a bispecific molecule comprising an anti-TCRVb antibody (Molecule H),an isotype control (BGM0122) or an anti-CD3e antibody (SP34), each at100 nM. Supernatant was collected on Days 1, 2, 3, 4, 5, 6, 7 and 8followed by multiplex analysis for the indicated cytokines orchemokines. The data was quantified using MSD (Meso Scale Discovery)platform, following the manufacturer's protocol. BGM0122 comprises theamino acid sequence of

(SEQ ID NO: 3283) METDTLLLWVLLLWVPGSTGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGLNDIFEAQKIEWHE.

FIGS. 25A-25J, FIGS. 26A-2611, and FIGS. 27A-27L show the levels ofcytokine and chemokine from PBMCs activated with the indicatedantibodies.

As shown in FIG. 25A, when plate-bound anti-TCR Vβ antibodies oranti-CD3e antibodies (OKT3) were used to activate human PBMCs, the Tcell cytokine IFNg was induced. With respect to IL-2 production, PBMCsactivated with anti-TCR Vβ antibodies resulted in increased IL-2production with delayed kinetics (FIG. 25B) as compared to PBMCsactivated with anti-CD3e antibody (OKT3).

While IL-1beta (FIG. 25C), IL-6 (FIG. 25D), IL-10 (FIG. 25E), IL-4 (FIG.25F), TNFalpha (FIG. 25G), IP-10 (FIG. 26C), IL-12-23p40 (FIG. 27D),IL-17A (FIG. 27G), and IL-1a (FIG. 2711), were induced by anti-CD3eantibody (OKT3), no or little induction of these cytokines or chemokineswas observed with PBMCs activated with anti-TCRVb antibodies.

PBMCs activated with anti-TCR Vβ antibodies demonstrated induction ofIL-13 (FIG. 251), IL-8 (FIG. 25J), Eotaxin (FIG. 26A), Eotaxin 3 (FIG.26B), IL-18 (HA) (FIG. 26C), MCP-1 (FIG. 26E), MCP-4 (FIG. 26F), MDC(FIG. 26G), MIP1a (FIG. 2611), MIP1B (FIG. 27A), TARC (FIG. 27B), GM-CSF(FIG. 27C), IL-15 (FIG. 27E), IL-16 (FIG. 27F), and IL-15 (FIG. 271),IL-7 (FIG. 27J).

Example 11: Nanostring-Based Gene Expression Profiling of TCRVb-Activated T Cells

This Example describes gene expression profiling of TCR Vβ-activated Tcells to, e.g., uncover potential mechanisms or pathways underlying TCRVβ activation of T cells.

In a first study, the anti-TCR Vβ13.1 antibody A-H.1 was compared withan anti-CD3 antibody OKT3. Briefly, human PBMCs were isolated from wholeblood. From isolated PBMCs, human CD3+ T cells were isolated usingmagnetic-bead separation (negative selection) (Miltenyi biotec) andactivated by immobilized (plate-coated) anti-TCR Vβ13.1 antibody (A-H.1)or anti-CD3 antibody (OKT3) at 100 nM for 6 days. Activated T-cells(from plate-coated) were then prepared for gene expression profiling(PanCancer IO 360™ Panel, nanoString), following manufacturer'sprotocol. Differential gene expression analysis was grouped by anti-TCRVβ13.1 (A-H.1) vs anti-CD3 (OKT3) activated T-cells using the nSolverAnalysis Software (Nanostring). Data shown in Table 15A are mean valuesfrom 3 donors. The differentially regulated genes shown in Table 15Ahave a p-value of 0.05 or less. In the fourth column of Table 15Ashowing fold changes in gene expression, a positive value indicatesgenes that are upregulated at the transcriptional level in TCRVβ-activated T cells compared to OKT3-activated T cells, whereas anegative value indicates genes downregulated at the transcriptionallevel in TCR Vβ-activated T cells compared to OKT3-activated T cells.

TABLE 15A Summary of genes whose expression are preferentially regulatedin TCR Vβ-activated T cells compared to OKT3-activated T cells. TCRVβ13.1 vs Probe Name Accession # NS Probe ID OKT3 Fold Change P valueCCR2 NM_001123041.2 NM_001123041.2: 743 −3.06 0.00019145 LIF NM_002309.3NM_002309.3: 1240 21.6 0.0003319 TCF7 NM_003202.2 NM_003202.2: 2420−8.38 0.00037035 PLA2G6 NM_001004426.1 NM_001004426.1: 1954 −2.190.00043564 CD84 NM_001184879.1 NM_001184879.1: 28 −3.81 0.00062413 ITGB2NM_000211.2 NM_000211.2: 520 −2.11 0.0012003 GZMK NM_002104.2NM_002104.2: 700 −11.09 0.00135083 HLA-DRB4 NM_021983.4 NM_021983.4: 194−5.75 0.00137591 CCR7 NM_001838.2 NM_001838.2: 1610 −2.43 0.00165716PDCD1 NM_005018.1 NM_005018.1: 175 7.24 0.00195468 CD96 NM_005816.4NM_005816.4: 1355 −6.44 0.00221401 SELL NR_029467.1 NR_029467.1: 1585 −50.00227156 NFATC4 NM_001136022.2 NM_001136022.2: 2296 −2.75 0.0025171CD8B NM_004931.3 NM_004931.3: 440 −3.56 0.00302475 NLRC5 NM_032206.4NM_032206.4: 860 −2.27 0.00309164 CD1C NM_001765.2 NM_001765.2: 750 8.620.0035729 HLA-B NM_005514.6 NM_005514.6: 937 −1.81 0.00363669 NUP107NM_020401.2 NM_020401.2: 1002 1.64 0.00366886 CD3D NM_000732.4NM_000732.4: 110 −2.05 0.00401569 HDAC3 NM_003883.2 NM_003883.2: 1455−1.41 0.0042794 PRKCE NM_005400.2 NM_005400.2: 1695 −1.86 0.00429076HLA-DQB1 NM_002123.3 NM_002123.3: 384 −5.71 0.00430297 AKT3 NM_181690.1NM_181690.1: 755 −2.98 0.00430433 VCAM1 NM_001078.3 NM_001078.3: 2535−23.93 0.00464703 CD53 NM_001040033.1 NM_001040033.1: 835 −1.70.00507702 LRP1 NM_002332.2 NM_002332.2: 4240 −2.22 0.00508974 CD28NM_001243078.1 NM_001243078.1: 2065 −1.73 0.00545641 OSM NM_020530.4NM_020530.4: 580 8.97 0.00558554 CLEC4A NM_194448.2 NM_194448.2: 388−1.7 0.0056661 MFGE8 NM_001114614.1 NM_001114614.1: 328 −2.75 0.00633707IFNAR2 NM_000874.3 NM_000874.3: 631 −3.69 0.00659279 LTA NM_000595.2NM_000595.2: 885 6.53 0.00727884 ITGAE NM_002208.4 NM_002208.4: 3405−3.42 0.00779862 CXCR5 NM_001716.3 NM_001716.3: 2618 4.38 0.00781195 CD6NM_006725.3 NM_006725.3: 1280 −1.38 0.00848703 ICOS NM_012092.2NM_012092.2: 640 1.74 0.00914866 NOS2A NM_153292.1 NM_153292.1: 546−2.29 0.0095337 CD1A NM_001763.2 NM_001763.2: 1815 5.12 0.00956367 CD27NM_001242.4 NM_001242.4: 330 −3.41 0.00984676 KLRD1 NM_002262.3NM_002262.3: 542 −6.43 0.00998325 TARP NM_001003799.1 NM_001003799.1:560 −3.71 0.00998698 HLA-DPB1 NM_002121.4 NM_002121.4: 931 −8.850.01064161 PTPRC NM_080921.3 NM_080921.3: 258 −2.86 0.01124117 CD44NM_001001392.1 NM_001001392.1: 429 −2.07 0.01138242 SLAMF6NM_001184714.1 NM_001184714.1: 1032 −1.81 0.00175123 HLA-DMB NM_002118.3NM_002118.3: 20 −6.39 0.01184625 CD276 NM_001024736.1 NM_001024736.1:2120 6.22 0.01207813 MAGEA1 NM_004988.4 NM_004988.4: 476 −2.930.01210408 HLA-DMA NM_006120.3 NM_006120.3: 380 −5.75 0.1210789 EP300NM_001429.2 NM_001429.2: 715 −1.24 0.01228626 ADA NM_000022.2NM_000022.2: 1300 −2.97 0.01228787 ICAM1 NM_000201.2 NM_000201.2: 22532.52 0.01290081 SIGIRR NM_021805.2 NM_021805.2: 469 −4.46 0.01309473 TNFNM_000594.2 NM_000594.2: 1010 4.6 0.01318389 IL1RAP NM_002182.2NM_002182.2: 460 2.77 0.01329693 CSF1 NM_000757.4 NM_000757.4: 823 2.550.01373637 CD40LG NM_000074.2 NM_000074.2: 1225 11.92 0.01376174 CYFIP2NM_001037332.2 NM_001037332.2: 4043 −1.38 0.01389707 MUC1 NM_001018017.1NM_001018017.1: 725 3.12 0.01399543 HLA-DRB3 NM_022555.3 NM_022555.3:698 −7.11 0.01404049 CD2 NM_001767.3 NM_001767.3: 687 −1.53 0.01432842IL2RG NM_000206.1 NM_000206.1: 595 −1.82 0.01477006 HLA-A NM_002116.5NM_002116.5: 1000 −1.96 0.01454336 TXK NM_003328.1 NM_003328.1: 800 −2.70.01590341 ITGA4 NM_000885.4 NM_000885.4: 975 −3.59 0.01601785 DHX16NM_001164239.1 NM_001164239.1: 2490 1.41 0.0167432 CD3E NM_000733.2NM_000733.2: 75 −1.52 0.01736902 MR1 NM_001531.2 NM_001531.2: 7695 −2.260.01744764 SMAD3 NM_005902.3 NM_005902.3: 4220 −2.82 0.01751245 CCRL2NM_003965.4 NM_003965.4: 1110 −1.87 0.01834479 HRAS NM_005343.2NM_005343.2: 396 1.97 0.0187379 IL18R1 NM_003855.2 NM_003855.2: 20252.36 0.01896204 CMA1 NM_001836.2 NM_001836.2: 561 −1.96 0.01964938 PSMB7NM_002799.2 NM_002799.2: 420 1.53 0.01980367 BCL10 NM_003921.2NM_003921.2: 1250 −1.38 0.01981376 HLA-DRA NM_019111.3 NM_019111.3: 335−7.46 0.02026993 CD80 NM_005191.3 NM_005191.3: 1288 4.18 0.02055337PIK3CD NM_005026.3 NM_005026.3: 2978 −1.23 0.02056576 ETS1 NM_005238.3NM_005238.3: 4625 −1.51 0.02083359 CHUK NM_001278.3 NM_001278.3: 8601.67 0.0217326 CCL5 NM_002985.2 NM_002985.2: 280 −2.47 0.02195802 ITGALNM_002209.2 NM_002209.2: 3905 −3 0.02244779 TNFRSF18 NM_004195.2NM_004195.2: 445 −3.76 0.02330885 EIF2B4 NM_172195.3 NM_172195.3: 13901.28 0.02349098 CD79A NM_001783.3 NM_001783.3: 695 −4.47 0.02361746ABCF1 NM_001090.2 NM_001090.2: 850 1.31 0.02452054 CD37 NM_001774.2NM_001774.2: 535 −2.06 0.02476513 STAT5B NM_012448.3 NM_012448.3: 200−1.56 0.02495121 CSF2 NM_000758.2 NM_000758.2: 475 11.38 0.0256982 STAT3NM_139276.2 NM_139276.2: 4535 −1.47 0.02629936 GZMA NM_006144.2NM_006144.2: 155 −2.46 0.02646368 C1R NM_001733.4 NM_001733.4: 760 −3.10.02653879 MIF NM_002415.1 NM_002415.1: 319 −1.38 0.02690018 CD46NM_172350.1 NM_172350.1: 365 −1.36 0.02725208 PIK3CG NM_002649.2NM_002649.2: 2125 −2.34 0.02762105 CFB NM_001710.5 NM_001710.5: 2029−2.59 0.02802998 IL3 NM_000588.3 NM_000588.3: 130 13.37 0.02820076TNFRSF13C NM_052945.3 NM_052945.3: 789 −2.2 0.02835259 MRPS5 NM_031902.3NM_031902.3: 390 1.2 0.02849936 TUBB NM_178014.2 NM_178014.2: 320 1.060.02874459 PECAM1 NM_000442.3 NM_000442.3: 1365 −4.35 0.02901845 PVRNM_006505.3 NM_006505.3: 604 2.28 0.0299334 AMICA1 NM_153206.2NM_153206.2: 620 −2.38 0.03034954 CD74 NM_001025159.1 NM_001025159.1:964 −3.28 0.0305419 ENTPD1 NM_001098175.1 NM_001098175.1: 8830 −8.020.03085618 CD97 NM_078481.2 NM_078481.2: 1370 −1.56 0.03086014 KLRK1NM_007360.3 NM_007360.3: 522 −4.16 0.03108504 HLA-DQA1 NM_002122.3NM_002122.3: 261 −5.51 0.03126291 CD247 NM_198053.1 NM_198053.1: 1490−1.88 0.03182703 IFNG NM_000619.2 NM_000619.2: 970 5.98 0.03202586 SAA1NM_199161.1 NM_199161.1: 135 −2.35 0.03341258 TBX21 NM_013351.1NM_013351.1: 890 1.92 0.03359165 RORA NM_134261.2 NM_134261.2: 1715−2.57 0.03591525 MASP2 NM_139208.1 NM_139208.1: 330 −1.65 0.03611762 CLUNM_001831.2 NM_001831.2: 2340 −1.55 0.0369776 KLRB1 NM_002258.2NM_002258.2: 85 −7.43 0.03705134 RELA NM_021975.2 NM_021975.2: 360 −1.260.03765981 SLAMF1 NM_003037.2 NM_003037.2: 580 1.82 0.03768168 CD8ANM_001768.5 NM_001768.5: 1320 −4.49 0.0380276 IL11RA NM_147162.1NM_147162.1: 400 −3.54 0.03855863 CD3G NM_000073.2 NM_000073.2: 404−1.44 0.03877635 JAK1 NM_002227.1 NM_002227.1: 285 −1.84 0.4001383 SPNNM_003123.3 NM_003123.3: 2345 −1.72 0.04035383 CXCR4 NM_003467.2NM_003467.2: 1335 −3.03 0.04122601 FAS NM_000043.3 NM_000043.3: 90 −2.370.04150638 IL2 NM_000586.2 NM_000586.2: 300 10.9 0.04175377 ITGA1NM_181501.1 NM_181501.1: 1875 −2.75 0.04213304 IGF1R NM_000875.2NM_000875.2: 455 −1.94 0.0424234 CLEC6A NM_001007033.1 NM_001007033.1:342 −2.83 0.04299769 RPS6 NM_001010.2 NM_001010.2: 171 −1.36 0.04334091MAPK11 NM_002751.5 NM_002751.5: 1310 −1.98 0.04344288 REL NM_002908.2NM_002908.2: 225 −2.37 0.04382344 EOMES NM_005442.2 NM_005442.2: 1670−6.49 0.04442535 KLRG1 NM_005810.3 NM_005810.3: 65 −3.52 0.04487411IL2RA NM_000417.1 NM_000417.1: 1000 3.4 0.0457568 IFNA17 NM_021268.2NM_021268.2: 291 −3.13 0.04595868 SH2D1B NM_053282.4 NM_053282.4: 545−1.44 0.04640447 CCL2 NM_002982.3 NM_002982.3: 123 4.01 0.04660539 TXNIPNM_006472.1 NM_006472.1: 255 −4.07 0.04695375 CXCL13 NM_006419.2NM_006419.2: 210 −65.05 0.04708191 CASP8 NM_001228.4 NM_001228.4: 301−1.42 0.04720592 MTMR14 NM_022485.3 NM_022485.3: 720 −1.25 0.04798024MAP3K5 NM_005923.3 NM_005923.3: 1760 −1.62 0.04838454 ADORA2ANM_000675.3 NM_000675.3: 1095 1.3 0.04872028 CCR5 NM_000579.1NM_000579.1: 2730 −4.01 0.04885927

In a second study, the multispecific anti-TCR Vβ13.1/anti-BCMA antibodyMolecule H was compared with the anti-CD3 antibody OKT3. Purified Tcells were stimulated with solid-phase anti-TCR Vβ antibody over 6 dayswith the anti-TCR Vβ antibody Molecule H or anti-CD3e antibody (OKT3) at100 nM. Expanded T cells were collected by centrifugation followed byRNA extraction. Seven hundred and seventy eight (778) immunology-relatedgenes were counted using the nCounter Technology (Nanostring) followedby gene expression analysis using nSolver analysis tools. The datadescribed in this Example is representative of 3 donors.

Based on this analysis, a panel of genes were identified as beingdifferentially regulated in TCR Vβ-activated T cells compared toOKT3-activated T cells (Table 15B). The differentially regulated genesshown in Table 15B have a p-value of 0.05 or less. For example, LIF,CD40LG, PDCD1, CXCR5, LTA, and CD80 are all upregulated at thetranscriptional level in TCR Vβ-activated T cells compared toOKT3-activated T cells. GZMK, ENTPD1 (CD39), TCF7, CD96, HLA-DRB4,SIGIRR and SELL are downregulated at the transcriptional level in TCRVβ-activated T cells compared to OKT3-activated T cells. TCRVβ-activated T cells also expressed high levels of cytolytic effectors(e.g., IFNg, Granzyme B and perforin).

TABLE 15B Summary of genes whose expression are preferentially regulatedin TCR Vβ-activated T cells compared to OKT3-activated T cells. Log₂Fold Gene Description Change P-Value LIF LIF Interleukin 6 FamilyCytokine 4.65 0.0119 GZMK Granzyme K −3.65 0.0468 CD40LG CD40 Ligand3.56 0.0082 ENTPD1 Ectonucleoside Triphosphate −3.53 0.0541 (CD39)Diphosphohydrolase 1 PDCD1 Programmed Cell Death 1 3.19 0.0257 TCF7Transcription Factor 7 −3.1 0.00634 CXCR5 Chemokine receptor for CXCL133.05 0.0337 CD96 Transmembrane glycoprotein Ig −2.75 0.007 superfamilyreceptor, interacts with nectin and nectin-like proteins, includingCD155/polio virus receptor (PVR) LTA Lymphotoxin Alpha 2.67 0.0082HLA-DRB4 Major Histocompatibility Complex, −2.66 0.0377 Class II, DRBeta 4 CD80 T cell costimulatory molecule 2.58 0.0425 SIGIRR Single IgAnd TIR Domain −2.37 0.0227 Containing SELL Selection L −2.3 0.00634

Example 12: Binding Affinity of Affinity Matured Humanized Antibody A-HAntibodies

This Example describes the evaluation of binding affinity of affinitymatured humanized Antibody A-H antibodies to recombinant protein TCRVB6-5.

Antibody A-H humanized antibodies were affinity matured. The resultingaffinity matured antibodies were tested for their binding affinity toTCRVB 6-5 as described below.

TCRVB 6-5 at 5 ug/mL was immobilized on a Biotin CAP Series S SensorChip to 60 RU. BJM0277 was diluted to 200 nM and then serially dilutedtwo fold. Association was 120 seconds, and dissociation was 300 seconds.This assay was run in 1×HBS-EP+ Buffer pH 7.4 and 25 C. The data was fitusing a 1:1 binding model.

TCRVB 6-5 at 5 ug/mL was immobilized on a Biotin CAP Series S SensorChip to 60 RU. A-H.45 was diluted to 50 nM and then serially diluted twofold. Association was 120 seconds, and dissociation was 300 seconds.This assay was run in 1×HBS-EP+Buffer pH 7.4 and 25 C. The data was fitusing a 1:1 binding model. A-H.45 is an improved yeast clone (TCRvB/CD19bispecific) and contains a mutation (G to V) at the last residue inframework 3, just before HCDR3. The affinity is 35-fold greater than theBJM0277 (Table 16).

TCRVB 6-5 at 5 ug/mL was immobilized on a Biotin CAP Series S SensorChip to 60 RU. A-H.52 was diluted to 50 nM and then serially diluted twofold. Association was 120 seconds, dissociation was 300 seconds. Thisassay was run in 1×HBS-EP+Buffer pH 7.4 and 25 C. The data was fit usinga 1:1 binding model. A-H.52 is a phage clones and is a monovalent scFv.A-H.52 has two mutations on CDRH1. The affinity of A-H.52 is 20-foldgreater than BJM0277 (Table 16).

TCRVB 6-5 at 5 ug/mL was immobilized on a Biotin CAP Series S SensorChip to 60 RU. A-H.53 was diluted to 50 nM and then serially diluted twofold. Association was 120 seconds, dissociation was 300 seconds. Thisassay was run in 1×HBS-EP+Buffer pH 7.4 and 25C. The data was fit usinga 1:1 binding model. A-H.53 (phage clone) affinity is in the pM range(Table 16). The affinity of A-H.53 is 200-fold greater than BJM0277(Table 16).

TCRVB 6-5 at 5 ug/mL was immobilized on a Biotin CAP Series S SensorChip to 60 RU. A-H.54 was diluted to 50 nM and then serially diluted twofold. Association was 120 seconds, dissociation was 300 seconds. Thisassay was run in 1×HBS-EP+Buffer pH 7.4 and 25C. The data was fit usinga 1:1 binding model. A-H.54 (phage clone) affinity is 17-fold greaterthan BJM0277 (Table 16).

TABLE 16 Summary of affinity maturation of anti-TCRVb antibodiesConstruct Target: TCRVβ 6-5 BJM0277 35 nM A-H.45 1.08 Nm A-H.52 1.76 nMA-H.53 165 pM A-H.54 2.22 nM

Example 13: Therapeutic Efficacy of CD19/TCRvB Bispecific Molecules inSubcutaneous Human Tumor Xenograft Models

This Example demonstrates the in vivo efficacy of a CD19/TCRvBBispecific molecule in a subcutaneous human tumor animal model.

On day 1 of the study 1×10⁶ cells of the human cancer cell line Raji,stably expressing firefly luciferase (Raji-luc) were subcutaneouslyinjected in the right dorsal flank of female NOD/SCID/IL-2Rγnull (NSG)mice. On day 3, 10×10⁶ human PBMCs were transplanted into mice byinjection into the peritoneal cavity.

Antibody treatment started at day 10, when tumors had reached a meantumor volume (TV) of 80 mm³. Mean TV of each group was not statisticallydifferent from any other group at start of treatment. Mice were treatedwith 0.2 mg/kg, 1 mg/kg and 5 mg/kg of CD19/TCRvB bispecific moleculeevery three days for a total of 7 doses by intravenous bolus injection.

Tumor volume (TV) was measured every 3 days by calipers and progressevaluated by intergroup comparison of TV. Tumor growth inhibition T/C[%] was calculated as T/C[%]=100×(mean TV of analyzed group)/(mean TV ofvehicle group).

Results are shown in Table 17 and FIG. 28. Treatment with the CD19/TCRvBBispecific molecule inhibited tumor growth compared to vehicle controltreatment (FIG. 28). The results demonstrate that the CD19/TCRvBbispecific molecule inhibits tumor growth and has anti-tumor activity.

TABLE 17 Mean tumor volume and tumor growth inhibition (T/C) at days 10to 28. Dose group Data D 10 D 13 D 16 D 19 D 23 D 25 D 28 Vehicle TV(mm³) 84 241 566 802 1577 2161 2478 T/C[%] 100 100 100 100 100 100 1000.2 mg/kg CD19/TCRvB TV (mm³) 82 169 460 643 967 946 875 T/C[%] 98 70 8180 61 44 35 1 mg/kg CD19/TCRvB TV (mm³) 82 122 147 307 482 469 406T/C[%] 98 51 26 38 31 22 16 5 mg/kg CD19/TCRvB TV (mm³) 79 160 200 381510 409 382 94 66 35 48 32 19 15

Example 14: Therapeutic Efficacy of CD19/TCRvB Bispecific Molecules inHuman Tumor Xenograft Models

This Example demonstrates the in vivo efficacy of a CD19/TCRvBBispecific molecules in a xenograft animal model.

On day 1 of the study 10×10⁶ human PBMCs were transplanted intoNOD/SCID/IL-2Rγnull (NSG) mice by injection into the peritoneal cavity.

On day 7, 1×10⁶ cells of the human cancer cell line Raji, stablyexpressing firefly luciferase (Raji-luc) were intravenously injectedinto NOD/SCID/IL-2Rγnull (NSG) mice. Control animals were injected with10×10⁶ cells of the CD19 negative human cancer cell line K562 stablyexpressing firefly luciferase (K562-luc). These animals were used toassess specific killing ability of CD19/TCRvB molecules. Antibodytreatment started at day 16, when tumor engraftment had reached a meanbioluminescence flux level of 4×10⁷ photons/s. Mean Flux level of eachgroup was not statistically different from any other group at start oftreatment. Mice were treated with 1 mg/kg and 5 mg/kg of CD19/TCRvBbispecific molecule every three days for a total of 6 doses byintravenous bolus injection.

Tumor burden was measured weekly by bioluminescence imaging and progressevaluated by intergroup comparison of total bioluminescence flux (TotalFlux). Tumor growth inhibition T/C [%] was calculated asT/C[%]=100×(mean Total Flux of analyzed group)/(mean Total Flux ofvehicle group).

The results for Raji-luc engrafted animals are shown in Table 18 andFIG. 29A and results for K562-luc engrafted animals are shown in Table19 and FIG. 29B. The results demonstrate that the CD19/TCRvB bispecificmolecule inhibits tumor growth and has anti-tumor activity (FIG. 29A andTable 18).

TABLE 18 Mean tumor burden (Total Flux) and tumor growth inhibition(T/C) at days 16 to 37 in animals engrafted with Raji-luc cells Dosegroup Data D 16 D 23 D 30 D 37 Vehicle Total Flux (p/s) 4.26E+075.92E+07 5.77E+08 4.23E+09 T/C[%] 100   100   100    100   1 mg/kg TotalFlux (p/s) 4.05E+07 2.66E+07 5.03E+07 5.42E+08 CD19/TCRvB T/C[%] 95.044.9 8.7 12.8 5 mg/kg Total Flux (p/s) 4.18E+07 3.10E+07 2.37E+071.44E+08 CD19/TCRvB T/C[%] 98.0 52.3 4.1  3.4

TABLE 19 Mean tumor burden (Total Flux) and tumor growth inhibition(T/C) at days 16 to 30 in animals engrafted with K562-luc cells Dosegroup Data D 16 D 23 D 30 Vehicle Total Flux (p/s) 2.98E+07 9.94E+082.40E+10 T/C[%] 100   100   100   5 mg/kg CD19/ Total Flux (p/s)2.00E+07 1.22E+09 3.82E+10 TCRvB T/C[%] 67.0 122.4 159.4

Example 15: Therapeutic Efficacy of BCMA/TCRvB Bispecific Molecules inHuman Tumor Xenograft Models

This Example demonstrates the in vivo efficacy of a BCMA/TCRvBBispecific molecule in a xenograft animal model.

On day 1, 20×10⁶ cells of the human cancer cell line RPMI-8226, stablyexpressing firefly luciferase (RPMI-8226-luc) were intravenouslyinjected into NOD/SCID/IL-2Rγnull (NSG) mice. On day 11, 10×10⁶ humanPBMCs were transplanted into mice by injection into the peritonealcavity. Antibody treatment started at day 17, when tumor engraftment hadreached a mean bioluminescence flux level of 4×10⁷ photons/s. Mice weretreated with 0.5 mg/kg of a molecule bivalent for both BCMA and TCRvB(2×2 molecule) and 0.5 mg/kg of a molecule bivalent for BCMA andmonovalent for TCRvB (2×1 molecule) once a week for a total of 2 dosesby intravenous bolus injection.

Tumor burden was measured weekly by bioluminescence imaging and progressevaluated by intergroup comparison of total bioluminescence flux (TotalFlux). Tumor growth inhibition T/C [%] was calculated asT/C[%]=100×(mean Total Flux of analyzed group)/(mean Total Flux ofvehicle group).

Results of these studies are shown in Table 20 and FIG. 30. Treatmentwith the BCMA/TCRvB Bispecific molecule inhibited tumor growth comparedto vehicle control treatment (FIG. 29). The results demonstrate that theBCMA/TCRvB bispecific molecule inhibits tumor growth and has anti-tumoractivity.

TABLE 20 Mean tumor burden (Total Flux) and tumor growth inhibition(T/C) at days 16 to 30. Dose group Data D 16 D 23 D 30 Vehicle TotalFlux (p/s) 3.71E+06 6.04E+06 7.29E+06 T/C[%] 100   100   100   0.5 mg/kgBCMA/ Total Flux (p/s) 7.33E+06 6.30E+06 1.13E+06 TCRvB 2 × 2 T/C[%]197.7 104.3 15.5 0.5mg/kg BCMA/ Total Flux (p/s) 3.66E+06 3.15E+065.65E+05 TCRvB 2 × 1 T/C[%]  98.8  52.1  7.8

Example 16: Expression and Purification of Antibody Constructs

Construction of the Plasmids

The DNA Encoding the Protein Sequences was Optimized for Expression inCricetulus griseus, synthesized, and cloned into the pcDNA3.4-TOPO (LifeTechnologies A14697) using Gateway cloning. All constructs contained anIg Kappa leader sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 3288).

Expression and Purification

The plasmids were co-transfected into either Expi293 cells (LifeTechnologies A14527) or ExpiCHO cells (Life Technologies A29127).Transfections were performed using 1 mg of total DNA for a multispecific construct with a 1:1 heavy chain ratio and 3:2 light chain toheavy chain ratio if applicable. Transfection in Expi293 cells was doneusing linear 25,000 Da polyethylenimine (PEI, Polysciences Inc 23966) ina 3:1 ratio with the total DNA. The DNA and PEI were each added to 50 mLof OptiMem (Life Technologies 31985088) medium and sterile filtered. TheDNA and PEI were combined for 10 minutes and added to the Expi293 cellswith a cell density of 1.8-2.8×10⁶ cells/mL and a viability of at least95%. The ExpiCHO transfection was performed according to themanufacturer's instructions. Expi293 cells were grown in a humidifiedincubator at 37° C. with 8% CO2 for 5-7 days after transfection andExpiCHO cells were grown for 14 days at 32° C. with 5% CO2. The cellswere pelleted by centrifugation at 4500×g and the supernatant wasfiltered through a 0.2 μm membrane. Protein A resin (GE 17-1279-03) wasadded to the filtered supernatant and incubated for 1-3 hours at roomtemperature. The resin was packed into a column, washed with 3 ×10column volumes of Dulbecco's phosphate-buffered saline (DPBS, LifeTechnologies 14190-144). The bound protein was eluted from the columnwith 20 mM citrate, 100 mM NaCl, pH 2.9. When necessary, the proteinswere further purified using ligand affinity and/or size exclusionchromatography on a Superdex 200 column with a running buffer of DPBS.

Example 17: Humanization of Anti-TRBV5-5 Antibody Clone Antibody C

The germline for the mouse anti-TCRvbeta antibody clone Antibody C VHand VL were assigned using IMGT nomenclature, with CDR regions definedby a combined Kabat and Chothia classification. SEQ ID NO: 232 and SEQID NO: 233 are the Antibody C VH and VL sequences respectively where theVH germline is mouse IGHV2-6-7*01 and the VL germline is mouseIGKV10-94*02. The method applied to humanize Antibody A described inExample 1 was used to humanize Antibody C. The Antibody C VH washumanized into human IGHV2-26*01, IGHV2-70*04, IGHV4-4*02, IGHV2-5*09,IGHV2-5*08, IGHV4-34*09, IGHV4-59*01, IGHV4-59*07, IGHV4-61*02,IGHV4-38-2*01, IGHV4-31*01, IGHV3-49*04, IGHV3-49*02, IGHV4-4*07,IGHV3-49*05, IGHV4-34*10, IGHV4-28*04, IGHV3-72*01, IGHV3-15*07,IGHV6-1*01, IGHV3-7*01, IGHV4-34*01, IGHV3-33*02, IGHV3-48*02,IGHV3-23*03, IGHV3-21*01, IGHV3-73*01, IGHV3-30*02, IGHV3-7*01,IGHV3-43*01, and IGHV3-53*03 and the Antibody C VL was humanized intohuman IGKV1D-43*01, IGKV1-27*01, IGKV1-17*02, IGKV1-17*01, IGKV1-5*01,IGKV4-1*01, IGKV3-7*02, IGKV3-7*01, IGKV2-29*02, IGKV6D-41*01,IGKV2-28*01, IGKV2-40*01, IGKV3-15*01, IGKV2-24*01, IGKV6-21*01,IGKV2D-26*01, and IGKV2D-26*03.

SEQ ID NOs: 3040-3089 are the Antibody C humanized heavy chains and SEQID NOs: 3000-3039 are the Antibody C humanized light chains (asdescribed in Table 10).

Example 18: Humanization of TRBV10-1, TRBV10-2, and TRBV10-3 AntibodyClone Antibody D

The germline for the mouse anti-TCRvbeta antibody clone Antibody D VHand VL were assigned using IMGT nomenclature, with CDR regions definedby a combined Kabat and Chothia classification. SEQ ID NO: 3183 and SEQID NO: 3184 are the Antibody D VH and VL sequences respectively wherethe VH germline is mouse IGHV5-6*01 and the VL germline is mouseIGKV4-59*01.

The method applied to humanize Antibody A described in Example 1 wasused to humanize Antibody D. The Antibody D VH was humanized into humanIGHV3-30*03, IGHV3-30*02, IGHV3-7*01, IGHV3-21*01, IGHV3-23*04,IGHV3-30*15, IGHV3-48*02, IGHV3-53*04, IGHV3-23*03, IGHV3-53*03,IGHV3-53*01, IGHV3-9*01, IGHV3-30*13, IGHV3-20*01, IGHV3-43D*03,IGHV3-43*02, IGHV3-43*01, IGHV3-53*02, IGHV3-13*01, IGHV3-38-3*01,IGHV3-9*03, IGHV3-64D*06, IGHV3-33*02, IGHV3-11*03, IGHV3-64*02,IGHV3-64*01, IGHV3-64*03, IGHV3-7*01, IGHV3-35*01, IGHV3-13*02,IGHV3-38*02, and IGHV3-38*01 and the Antibody D VL was humanized intohuman IGKV3-11*01, IGKV1-13*02, IGKV1-9*01, IGKV6-21*01, IGKV1D-43*01,IGKV3-11*01, IGKV3D-11*02, IGKV1-17*03, IGKV3D-20*01, IGKV3-20*01,IGKV1D-16*01, IGKV4-1*01, IGKV2-28*01, IGKV2-40*01, IGKV2-29*02,IGKV2-29*01, IGKV1D-42*01, IGKV2-24*01, and IGKV5-2*01. SEQ ID NOs:3225-3274 are the Antibody D humanized heavy chains and SEQ ID NOs:3185-3224 are the Antibody D humanized light chains (as described inTable 12).

Example 19: Humanization of TRBV5-5 and TRBV5-6 Antibody Clone AntibodyE

The germline for the mouse anti-TCRβ antibody clone Antibody E VH and VLwere assigned using IMGT nomenclature, with CDR regions defined by acombined Kabat and Chothia classification. SEQ ID NO: 3091 and SEQ IDNO: 3092 are the Antibody E VH and VL sequences respectively where theVH germline is mouse IGHV1-82*01 and the VL germline is mouseIGKV3-5*01.

The method applied to humanize Antibody A described in Example 1 wasused to humanize Antibody E. The Antibody E VH was humanized into humanIGHV1-69*08, IGHV1-3*02, IGHV1-18*03, IGHV1-3*01, IGHV1-18*01,IGHV1-2*06, IGHV1-2*01, IGHV1-2*06, IGHV1-8*01, IGHV7-4-1*02,IGHV1-58*02, IGHV5-51*01, IGHV7-4-1*04, IGHV7-81*01, IGHV5-51*04,IGHV5-51*01, IGHV1-45*03, IGHV3-49*04, IGHV3-49*02, IGHV3-49*05,IGHV4-4*02, IGHV3-49*05, IGHV3-73*01, IGHV4-4*02, IGHV3-15*07,IGHV3-15*02, IGHV3-72*01, IGHV4-59*07, IGHV4-31*01, IGHV4-31*02,IGHV3-30*15, IGHV3-21*01, IGHV3-7*01, IGHV4-28*01, IGHV4-28*02,IGHV3-30*08, IGHV3-30*05, and IGHV3-30*01 and the Antibody E VL washumanized into human IGKV4-1*01, IGKV3-11*01, IGKV3-20*02, IGKV3-11*01,IGKV1-13*02, IGKV3D-11*01, IGKV3D-20*02, IGKV1-13*02, IGKV3D-20*01,IGKV1-9*01, IGKV3D-15*03, IGKV3-15*01, IGKV1-5*01, IGKV2D-29*01,IGKV3-7*02, IGKV1-9*01, IGKV2-28*01, IGKV2-40*01, IGKV2D-29*02,IGKV3-7*01, IGKV2-30*01, IGKV2-24*01, IGKV6D-41*01, IGKV1D-42*01,IGKV2D-26*01, IGKV2D-26*03, and IGKV5-2*01. SEQ ID NOs: 3133-3182 arethe Antibody E humanized heavy chains and SEQ ID NOs: 3093-3132 are theAntibody E humanized light chains (as described in Table 11).

Example 20: In Vitro Cytotoxicity of an Anti-TCRVb/CD19 AntibodyMolecule and an Anti-TCRVb/BCMA Antibody Molecule

Anti-TCR/Anti-CD19 Dual Targeting Antibody Molecule

Human PBMCs were isolated from whole blood. From isolated PBMCs, humanCD3+ T cells were isolated using magnetic-bead separation (negativeselection) (Miltenyi biotec) and activated by immobilized (plate-coated)anti-TCR Vβ13.1 (A-H.1) at 100 nM for 6 days. Activated T-cells (fromplate-coated) were then transferred and expanded in tissue culture flaskin the presence of human IL-2 at a concentration of 50 U/ml for twoadditional days. Expanded TCR Vβ13.1+ cells were washed and co-culturedin the presence of CD19-expressing Raji cells (target cells) at an E:Tratio of 5:1 and serial diluted concentrations of T-cell engagerbispecific antibodies including, anti-TCR Vβ13.1/CD19 (Molecule F),anti-CD3/CD19, and anti-TCR Vβ13.1 (A-H.1, serving as control) for 24hours. Post 24 hours, cell co-culture supernatants were collected andquantified for specific target cell death. Target cells (Raji cells) area KILR-retroparticles reporter cell assay (DiscoverX). KILR-Raji targetcells are engineered to stably express a protein tagged with enhanced.ProLabel (ePL), a reporter fragment, using the MLR Retroparticles, andwhen the membrane of the target cells is compromised due to cell death,the target cells will release the tagged protein into the media. ThisKILR reporter protein can be detected in the media/supernatant by theaddition of detection reagents containing the enzyme acceptor (EA)fragment of the β-gal reporter. This leads to the formation of theactive β-gal enzyme which hydrolyzes the substrate to give achemiluminescent output (RLU). Percentage (%) of target cell death iscalculated using the following formula:

(RLU_(Treatment)−RLU_(No Treatment))/(RLU_(Maximum Lysis)−RLU_(No Treatment))×100

Data shown in FIG. 31A are mean values from 4 donors.

Anti-TCR/Anti-BCMA Dual Targeting Antibody Molecule

Human PBMCs were isolated from whole blood. From isolated PBMCs, humanCD3+ T cells were isolated using magnetic-bead separation (negativeselection) (Miltenyi biotec) and activated by immobilized (plate-coated)anti-TCR Vβ13.1 (A-H.1) at 100 nM for 6 days. Activated T-cells (fromplate-coated) were then transferred and expanded in tissue culture flaskin the presence of human IL-2 at a concentration of 50 U/ml for twoadditional days. Expanded TCR Vβ13.1+ cells were washed and co-culturedin the presence of BCMA-expressing RPMI8226 cells (target cells) at anE:T ratio of 5:1 and serial diluted concentrations of T-cell engagerbispecific antibodies including, anti-TCR Vβ13.1/BCMA (Molecule G),anti-CD3/BCMA, and anti-TCR Vβ13.1 (A-H.1, serving as control) for 24hours. Post 24 hours, cell co-culture supernatants were collected andquantified for specific target cell death. Target cells (RPMI8226 cells)are a KILR-retroparticles reporter cell assay (DiscoverX). KILR-RPMI8226target cells are engineered to stably express a protein tagged withenhanced ProLabel (ePL), a β-gal reporter fragment, using the MLRRetroparticles, and when the membrane of the target cells is compromiseddue to cell death, the target cells will release the tagged protein intothe media. This Knit reporter protein was detected and percentage (%) oftarget cell death was calculated as described above. Data shown in FIG.31B are mean values from 4 donors.

Example 21: Cytokine Profile of an Anti-TCRVb/BCMA Antibody Molecule

This Examples describes cytokines secreted by PBMCs following activationby the anti-TCR Vβ/anti-BCMA antibody Molecule H. For comparison,activation by an anti-TCR beta constant 1 (TRBC1) antibody Antibody Fwas also analyzed.

Briefly, human PBMCs were isolated from whole blood followed bysolid-phase (plate-coated) stimulation with Molecule H or Antibody F at100 nM. Supernatant was collected on Days 1, 2, 3, and 5 (for MoleculeH) or Days 2 and 5 (for Antibody F) followed by multiplex cytokineanalysis for IFNγ, IL-2, IL-1β, IL-6, IL-10, and TNFα, quantified usingMSD (Meso Scale Discovery) platform, following the manufacturer'sprotocol.

As shown in FIGS. 32A-32F and 33A-33F, the cytokine profile of theanti-TCR Vβ/anti-BCMA antibody Molecule H is different from that of theanti-CD3 antibody OKT3 or the anti-TRBC1 Antibody F.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

EXEMPLARY EMBODIMENTS

Disclosed herein are, inter alia, antibody molecules directed to thevariable chain of the beta subunit of TCR (TCRβV) which bind and, e.g.,activate or expand, T cells, e.g., a subset of T cells (“anti-TCRβVantibody molecules”). In some embodiments, the anti-TCRβV antibodymolecules disclosed herein result in a cytokine profile, e.g., acytokine secretion profile, that differs from that of a T cell engagerthat binds to a receptor or molecule other than a TCRβV region (“anon-TCRβV-binding T cell engager”). In some embodiments, the anti-TCRβVantibody molecules disclosed herein result in lesser, minimal, or noproduction of cytokines associated with cytokine release syndrome (CRS),e.g., IL-6, IL-1beta, IL-10 and TNF alpha; and enhanced and/or delayedproduction of IL-2 and IFN-gamma. In some embodiments, the anti-TCRβVantibodies disclosed herein result in expansion of an immune cell, e.g.,a T cell, a tumor infiltrating lymphocyte (TIL), an NK cell, or otherimmune cells (e.g., as described herein). Also provided herein aremethods of making said anti-TCRβV antibody molecules, and methods ofusing said anti-TCRβV antibody molecules including, methods of using ananti-TCRβV antibody molecule for expanding an immune cell or an immunecell population, and method of using an anti-TCRβV antibody molecule fortreating cancer, including the use as combination therapy with TIL andimmune checkpoint therapeutics. This disclosure further providesmultispecific molecules, e.g., bispecific molecules, comprising saidanti-TCRβV antibody molecules. In some embodiments, compositionscomprising anti-TCRβV antibody molecules of the present disclosure, canbe used, e.g., to activate and/or redirect T cells to promote tumor celllysis for cancer immunotherapy. In some embodiments, compositionscomprising anti-TCRβV antibody molecules as disclosed herein limit theunwanted side-effects of CRS and/or NT, e.g., CRS and/or NT associatedwith anti-CD3e targeting.

In some embodiments, the anti-TCRβV antibody molecules disclosed hereinresult in lesser, minimal, or no production of cytokines associated withcytokine release syndrome (CRS), e.g., IL-6, IL-1beta, IL-10 and TNFalpha; and enhanced and/or delayed production of IL-2 and IFN-gamma,compared with an anti-CD3 antibody molecule (e.g., a low affinityanti-CD3 antibody molecule). In some embodiments, administration of theanti-TCRβV antibody molecules disclosed herein in a subject results inreduced cytokine release syndrome (CRS) (e.g., lesser duration of CRS orno CRS), a reduced severity of CRS (e.g., absence of severe CRS, e.g.,CRS grade 4 or 5), reduced neurotoxicity (NT), or a reduced severity ofNT, compared with similar administration of an anti-CD3 antibodymolecule (e.g., a low affinity anti-CD3 antibody molecule).

Accordingly, provided herein are, anti-TCRβV antibody molecules,multispecific or multifunctional molecules (e.g., multispecific ormultifunctional antibody molecules) (also referred to herein as a“composition”) that comprise anti-TCRβV antibody molecules, nucleicacids encoding the same, methods of producing the aforesaid molecules,pharmaceutical compositions comprising aforesaid molecules, and methodsof treating a disease or disorder, e.g., cancer, using the aforesaidmolecules. The antibody molecules and pharmaceutical compositionsdisclosed herein can be used (alone or in combination with other agentsor therapeutic modalities) to treat, prevent and/or diagnose disordersand conditions, e.g., cancer, e.g., as described herein.

In one aspect, the disclosure provides an antibody molecule, e.g., anon-murine, e.g., a human-like (e.g., a human, or humanized antibodymolecule), which binds, e.g., specifically binds, to a T cell receptorbeta variable (TCRβV) region.

In some embodiments, the anti-TCRBV antibody molecule comprises anantigen binding domain of an antibody disclosed in any of Tables 1-2, or10-12, or a sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%identity thereto. In some embodiments, the anti-TCRBV antibody moleculecomprises a leader sequence comprising the amino acid sequence of SEQ IDNO: 3288. In some embodiments, the anti-TCRBV antibody molecule does notcomprise a leader sequence comprising the amino acid sequence of SEQ IDNO: 3288.

In some embodiments, binding of the anti-TCRβV antibody molecule to aTCRβV region results in a cytokine profile, e.g., a cytokine secretionprofile, (e.g., comprising one or more cytokines and/or one or morechemokines), that differs from that of a T cell engager that binds to areceptor or molecule other than a TCRβV region (“a non-TCRβV-binding Tcell engager”).

In some embodiments, the cytokine profile, e.g., cytokine secretionprofile, comprises one, two, three, four, five, six, seven, or all ofthe following:

(i) increased level, e.g., expression level, and/or activity of IL-2;(ii) reduced level, e.g., expression level, and/or activity of IL-1β;(iii) reduced level, e.g., expression level, and/or activity of IL-6;(iv) reduced level, e.g., expression level, and/or activity of TNFα;(v) reduced level, e.g., expression level, and/or activity of IL-10;(vi) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hoursdelay, in increased level, e.g., expression level, and/or activity ofIL-2;(vii) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours delay,in increased level, e.g., expression level, and/or activity ofIFN-gamma; or(viii) increased level, e.g., expression level, and/or activity ofIL-15, e.g., wherein (i)-(viii) are relative to the cytokine profile,e.g., cytokine secretion profile, of the non-TCRβV-binding T cellengager.

In some embodiments, binding of the anti-TCRBV antibody to a TCRβVregion results in reduced cytokine storm, e.g., reduced cytokine releasesyndrome (CRS) and/or neurotoxicity (NT), as measured by an assay ofExample 3, e.g., relative to the cytokine storm induced by thenon-TCRβV-binding T cell engager.

In some embodiments, binding of the anti-TCRBV antibody to a TCRβVregion results in one, two, three or all of:

(ix) reduced T cell proliferation kinetics;(x) cell killing, e.g., target cell killing, e.g. cancer cell killing,e.g., as measured by an assay of Example 4;(xi) increased Natural Killer (NK) cell proliferation, e.g., expansion;or(xii) expansion, e.g., at least about 1.1-10 fold expansion (e.g., atleast about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 foldexpansion), of a population of memory T cells, e.g., wherein (ix)-(xii)are relative to the non-TCRBV-binding T cell engager.

In some embodiments, an anti-TCRβV antibody molecule disclosed hereinrecognizes (e.g., binds to), a structurally conserved domain on theTCRβV protein (e.g., as denoted by the circled area in FIG. 24A).

In some embodiments, an anti-TCRβV antibody molecule disclosed hereindoes not recognize, e.g., bind to, an interface of a TCRβV:TCRalphacomplex.

In some embodiments, an anti-TCRβV antibody molecule disclosed hereindoes not recognize, e.g., bind to, a constant region of a TCRβV protein.An exemplary antibody that binds to a constant region of a TCRBV regionis JOVI. 1 as described in Viney et al., (Hybridoma. 1992 December;11(6):701-13).

In some embodiments, an anti-TCRβV antibody molecule disclosed hereindoes not recognize, e.g., bind to, one or more (e.g., all) of acomplementarity determining region (e.g., CDR1, CDR2 and/or CDR3) of aTCRβV protein.

In some embodiments, binding of the anti-TCRβV antibody molecule to aTCRβV region results in one, two, three, four, five, six, seven, eight,nine, ten or more (e.g., all) of the following:

(i) reduced level, e.g., expression level, and/or activity of IL-1β;(ii) reduced level, e.g., expression level, and/or activity of IL-6;(iii) reduced level, e.g., expression level, and/or activity of TNFα;(iv) increased level, e.g., expression level, and/or activity of IL-2;(v) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hoursdelay, in increased level, e.g., expression level, and/or activity ofIL-2;(vi) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours delay,in increased level, e.g., expression level, and/or activity ofIFN-gamma;(vii) reduced T cell proliferation kinetics;(viii) reduced cytokine storm, e.g., cytokine release syndrome (CRS)and/or neurotoxicity (NT), e.g., as measured by an assay of Example 3;(ix) cell killing, e.g., target cell killing, e.g. cancer cell killing,e.g., as measured by an assay of Example 4;(x) increased level, e.g., expression level, and/or activity of IL-15;or(xi) increased Natural Killer (NK) cell proliferation, e.g., expansion.

In some embodiments, any one or all of (i)-(xi) or any combinationthereof resulting from an anti-TCRβV antibody molecule disclosed hereinis compared to an antibody that binds to: a CD3 molecule, e.g., CD3epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

In some embodiments, binding of the anti-TCRβV antibody molecule to aTCRβV region results in secretion, e.g., production of perforin and/orGranzyme B.

In an aspect, the disclosure provides an antibody molecule which binds,e.g., specifically binds, to a T cell receptor beta variable chain(TCRβV) region, wherein the anti-TCRβV antibody molecule comprises anantigen binding domain comprising:

(a) a light chain variable region (VL) comprising:

(i) one, two or all of (e.g., three) a light chain complementaritydetermining region 1 (LC CDR1), a light chain complementaritydetermining region 2 (LC CDR2), and a light chain complementaritydetermining region 3 (LC CDR3) of SEQ ID NO: 10 or SEQ ID NO: 11; and

(ii) a framework region (FR) having at least 95% identity with one, two,three, or all of (e.g., four) a non-murine germline framework 1 (FR1), anon-murine germline framework region 2 (FR2), a non-murine germlineframework region 3 (FR3), and a non-murine germline framework region 4(FR4); and/or

(b) a heavy chain variable region (VH) comprising:

(i) one, two or all of (e.g., three) a heavy chain complementaritydetermining region 1 (HC CDR1), a heavy chain complementaritydetermining region 2 (HC CDR2) and a heavy chain complementaritydetermining region 3 (HC CDR3) of SEQ ID NO: 9; and

(ii) a framework region (FR) having at least 95% identity with one, two,three, or all of (e.g., four) a non-murine germline framework 1 (FR1), anon-murine germline framework region 2 (FR2), a non-murine germlineframework region 3 (FR3), and a non-murine germline framework region 4(FR4).

In some embodiments, the VL comprises a sequence having a consensussequence of SEQ ID NO: 230 or 3289.

In some embodiments, the VH comprises a sequence having a consensussequence of SEQ ID NO: 231 or 3290.

In some embodiments, the anti-TCRβV antibody molecule binds to TCRβ V6,e.g., one or more of TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβV6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβV6-3*01 or TCRβ V6-1*01, or a variant thereof.

In some embodiment, the anti-TCRβV antibody molecule comprises anantigen binding domain comprising:

(i) a HC CDR1, a HC CDR2 and a HC CDR3 of SEQ ID NO: 1 or SEQ ID NO: 9,or an amino acid sequence listed in Table 1; or

(ii) a LC CDR1, a LC CDR2, and a LC CDR3 of SEQ ID NO: 2, SEQ ID NO: 10or SEQ ID NO: 11, or an amino acid sequence listed in Table 1.

In some embodiments, the anti-TCRβV antibody molecule comprises anantigen binding domain comprising a light chain variable region (VL)comprising one, two or all (e.g., three) of a LC CDR1, a LC CDR2 and aLC CDR3 of SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 11, or an aminoacid sequence listed in Table 1.

In some embodiments, the anti-TCRβV antibody molecule comprises anantigen binding domain comprising a heavy chain variable region (VH)comprising one, two or all (e.g., three) of a HC CDR1, a HC CDR2 and aHC CDR3 of SEQ ID NO:1 or SEQ ID NO: 9, or an amino acid sequence listedin Table 1.

In some embodiments, the anti-TCRβV antibody molecule comprises anantigen binding domain comprising:

(i) a VL comprising: a LC CDR1 amino acid sequence of SEQ ID NO: 6 (oran amino acid sequence with not more than 1, 2, 3 or 4 modifications,e.g., substitutions, additions or deletions thereof), a LC CDR2 aminoacid sequence of SEQ ID NO:7 (or an amino acid sequence with not morethan 1, 2, 3 or 4 modifications, e.g., substitutions, additions ordeletions thereof), and/or a LC CDR3 amino acid sequence of SEQ ID NO:8(or an amino acid sequence with not more than 1, 2, 3 or 4modifications, e.g., substitutions, additions or deletions thereof);and/or

(ii) a VH comprising: a HC CDR1 amino acid sequence of SEQ ID NO: 3 (oran amino acid sequence with not more than 1, 2, 3 or 4 modifications,e.g., substitutions, additions or deletions thereof), a HC CDR2 aminoacid sequence of SEQ ID NO:4 (or an amino acid sequence with not morethan 1, 2, 3 or 4 modifications, e.g., substitutions, additions ordeletions thereof), and/or a HC CDR3 amino acid sequence of SEQ ID NO:5(or an amino acid sequence with not more than 1, 2, 3 or 4modifications, e.g., substitutions, additions or deletions thereof).

In some embodiments, the anti-TCRβV antibody molecule comprises anantigen binding domain comprising:

a variable heavy chain (VH) of an amino acid sequence listed in Table 1,e.g., SEQ ID NO: 9, or a sequence having at least about 85%, 90%, 95%,or 99% sequence identity to an amino acid sequence listed in Table 1,e.g., SEQ ID NO: 9; and/or a variable light chain (VL) of an amino acidsequence listed in Table 1, e.g., SEQ ID NO: 10 or SEQ ID NO: 11, or asequence having at least about 85%, 90%, 95%, or 99% sequence identityto an amino acid sequence listed in Table 1, e.g., SEQ ID NO: 10 or SEQID NO: 11.

In some embodiments, the anti-TCRβV antibody molecule comprises anantigen binding domain comprising:

(i) the VH amino acid sequence of SEQ ID NO: 9;

(ii) an amino acid sequence having at least about 85%, 90%, 95%, or 99%sequence identity to the amino acid sequence of SEQ ID NO: 9;

(iii) the VL amino acid sequence of SEQ ID NO: 10; and/or

(iv) an amino acid sequence having at least about 85%, 90%, 95%, or 99%sequence identity to the amino acid sequence of SEQ ID NO: 10.

In an aspect, provided herein is an antibody molecule which binds, e.g.,specifically binds, to a T cell receptor beta variable chain (TCRβV)region, wherein the anti-TCRβV antibody molecule comprises an antigenbinding domain comprising:

(a) a light chain variable region (VL) comprising:

(i) one, two or all of (e.g., three) a light chain complementaritydetermining region 1 (LC CDR1), a light chain complementaritydetermining region 2 (LC CDR2), and a light chain complementaritydetermining region 3 (LC CDR3) of a humanized B-H light chain (LC) ofTable 2; and

(ii) a framework region (FR) having at least 95% identity with one, two,three or all (e.g., four) of a framework region 1 (FR1), a frameworkregion 2 (FR2), a framework region 3 (FR3), and a framework region 4(FR4) of a humanized B-H LC of Table 2; and/or

(b) a heavy chain variable region (VH) comprising:

(i) one, two or all of (e.g., three) a heavy chain complementaritydetermining region 1 (HC CDR1), a heavy chain complementaritydetermining region 2 (HC CDR2) and a heavy chain complementaritydetermining region 3 (HC CDR3) of a humanized B-H heavy chain (HC) ofTable 2; and

(ii) a framework region (FR) having at least 95% identity with one, two,three or all (e.g., four) of a framework region 1 (FR1), a frameworkregion 2 (FR2), a framework region 3 (FR3), and a framework region 4(FR4) of a humanized B-H HC of Table 2.

In some embodiments, the anti-TCRBV binds to TCRβ V12, e.g., TCRβV12-4*01, TCRβ V12-3*01, or TCRβ V12-5*01, or a variant thereof.

In some embodiment, the anti-TCRβV antibody molecule comprises anantigen binding domain comprising:

(i) a HC CDR1, a HC CDR2 and a HC CDR3 of Antibody B listed in Table 2;or

(ii) a LC CDR1, a LC CDR2, and a LC CDR3 of Antibody B listed in Table2.

In some embodiments, the anti-TCRβV antibody molecule comprises anantigen binding domain comprising a light chain variable region (VL)comprising one, two or all (e.g., three) of a LC CDR1, a LC CDR2 and aLC CDR3 of SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 11, or an aminoacid sequence listed in Table 1.

In some embodiments, the anti-TCRβV antibody molecule comprises anantigen binding domain comprising a heavy chain variable region (VH)comprising one, two or all (e.g., three) of a HC CDR1, a HC CDR2 and aHC CDR3 of a humanized B-H antibody listed in Table 2.

In some embodiments, the anti-TCRβV antibody molecule comprises anantigen binding domain comprising a light chain variable region (VL)comprising one, two or all (e.g., three) of a LC CDR1, a LC CDR2 and aLC CDR3 of a humanized B-H antibody listed in Table 2.

In some embodiments, the anti-TCRβV antibody molecule comprises:

a VH sequence of a humanized B-H antibody listed in Table 2, or asequence having at least about 85%, 90%, 95%, or 99% sequence identityto a VH of a humanized B-H antibody listed in Table 2; and/or

a VL sequence of a humanized B-H antibody listed in Table 2, or asequence having at least about 85%, 90%, 95%, or 99% sequence identityto a VL of a humanized B-H antibody listed in Table 2.

In some embodiments, the anti-TCRβV antibody molecule comprises aframework region (FR) having at least 95% identity with one of: a FR1, aFR2, a FR3, and a FR4 of a humanized B-H LC of Table 2.

In some embodiments, the anti-TCRβV antibody molecule comprises aframework region (FR) having at least 95% identity with any two of: aFR1, a FR2, a FR3, and a FR4 of a humanized B-H LC of Table 2.

In some embodiments, the anti-TCRβV antibody molecule comprises aframework region (FR) having at least 95% identity with any three of: aFR1, a FR2, a FR3, and a FR4 of a humanized B-H LC of Table 2.

In some embodiments, the anti-TCRβV antibody molecule comprises aframework region (FR) having at least 95% identity with all of: a FR1, aFR2, a FR3, and a FR4 of a humanized B-H LC of Table 2.

In some embodiments, the anti-TCRβV antibody molecule comprises aframework region (FR) having at least 95% identity with one of: a FR1, aFR2, a FR3, and a FR4 of a humanized B-H HC of Table 2.

In some embodiments, the anti-TCRβV antibody molecule comprises aframework region (FR) having at least 95% identity with any two of: aFR1, a FR2, a FR3, and a FR4 of a humanized B-H HC of Table 2.

In some embodiments, the anti-TCRβV antibody molecule comprises aframework region (FR) having at least 95% identity with any three of: aFR1, a FR2, a FR3, and a FR4 of a humanized B-H HC of Table 2.

In some embodiments, the anti-TCRβV antibody molecule comprises aframework region (FR) having at least 95% identity with all of: a FR1, aFR2, a FR3, and a FR4 of a humanized B-H HC of Table 2.

In another aspect, the disclosure provides a non-murine, e.g., ahuman-like antibody molecule (e.g., a human or humanized antibodymolecule), which binds, e.g., specifically binds, to a T cell receptorbeta variable (TCRβV) region. In some embodiments, binding of theanti-TCRβV antibody molecule results in expansion, e.g., at least about1.1-50 fold expansion (e.g., at least about 1.5-40 fold, 2-35 fold, 3-30fold, 5-25 fold, 8-20 fold, or 10-15 fold expansion), of a population ofmemory T cells, e.g., T effector memory (T_(EM)) cells, e.g., T_(EM)cells expressing CD45RA (T_(EMRA)) cells, e.g., CD4+ or CD8+ T_(EMRA)cells. In some embodiments, the expansion is at least about 1.1-10 foldexpansion (e.g., at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6,7, 8, 9, or 10 fold expansion).

In some embodiments, expansion of the population of memory effector Tcells, e.g., T_(EM) cells, e.g., T_(EMRA) cells, e.g., CD4+ or CD8+T_(EMRA) cells, is compared to expansion of a similar population ofcells with an antibody that binds to: a CD3 molecule, e.g., CD3 epsilon(CD3e) molecule; or a TCR alpha (TCRα) molecule.

In some embodiments, the population of expanded T effector memory cellscomprises cells T cells, e.g., CD3+, CD8+ or CD4+ T cells. In someembodiments, the population of expanded T effector memory cellscomprises CD3+ and CD8+ T cells. In some embodiments, the population ofexpanded T effector memory cells comprises CD3+ and CD4+ T cells.

In some embodiments, the population of expanded T effector memory(T_(EM)) cells comprises cells T cells, e.g., CD3+, CD8+ or CD4+ Tcells, which express or re-express, CD45RA, e.g., CD45RA+. In someembodiments, the population comprises T_(EM) cells expressing CD45RA,e.g., T_(EMRA) cells. In some embodiments, expression of CD45RA onT_(EMRA) cells, e.g., CD4+ or CD8+ T_(EMRA) cells, can be detected by amethod disclosed herein, e.g., flow cytometry.

In some embodiments, T_(EMRA) cells have low or no expression of CCR7,e.g., CCR7- or CCR7 low. In some embodiments, expression of CCR7 onT_(EMRA) cells cannot be detected by a method disclosed herein, e.g.,flow cytometry.

In some embodiments, T_(EMRA) cells express CD95, e.g., CD95+. In someembodiments, expression of CD95 on T_(EMRA) cells can be detected by amethod disclosed herein, e.g., flow cytometry.

In some embodiments, T_(EMRA) cells express CD45RA, e.g., CD45RA+, havelow or no expression of CCR7, e.g., CCR7− or CCR7 low, and express CD95,e.g., CD95+. In some embodiments T_(EMRA) cells can be identified asCD45RA+, CCR7− and CD95+ cells. In some embodiments, T_(EMRA) cellscomprise CD3+, CD4+ or CD8+ T cells (e.g., CD3+ T cells, CD3+CD8+ Tcells, or CD3+CD4+ T cells).

In some embodiments, binding of the anti-TCRβV antibody molecule resultsin expansion, e.g., at least about 1.1-50 fold expansion (e.g., at leastabout 1.5-40 fold, 2-35 fold, 3-30 fold, 5-25 fold, 8-20 fold, or 10-15fold expansion), of a subpopulation of T cells. In some embodiments, theanti-TCRβV antibody molecule-activated (e.g., expanded) subpopulation ofT cells resemble T_(EMRA) cells in high expression of CD45RA and/or lowexpression of CCR7. In some embodiments, the anti-TCRβV antibodymolecule-activated (e.g., expanded) subpopulation of T cells do notdisplay upregulation of the senescence markers CD57 and/or KLRG1. Insome embodiments, the anti-TCRβV antibody molecule-activated (e.g.,expanded) subpopulation of T cells do not display upregulation ofco-stimulatory molecules CD27 and/or CD28. In some embodiments, theanti-TCRβV antibody molecule-activated (e.g., expanded) subpopulation ofT cells are highly proliferative. In some embodiments, the anti-TCRβVantibody molecule-activated (e.g., expanded) subpopulation of T cellssecrete IL-2. In some embodiments, expression of surface markers on Tcells can be detected by a method disclosed herein, e.g., flowcytometry. In some embodiments, the proliferative capability of T cellscan be detected by a method disclosed herein, e.g., a method describedin Example 4. In some embodiments, cytokine expression of T cells can bedetected by a method disclosed herein, e.g., a method described inExamples 10 and 21. In some embodiments, the expansion is at least about1.1-10 fold expansion (e.g., at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2,3, 4, 5, 6, 7, 8, 9, or 10 fold expansion). In some embodiments, theexpansion is compared to expansion of a similar population of cells withan antibody that binds to a CD3 molecule, e.g., CD3 epsilon (CD3e)molecule; or a TCR alpha (TCRα) molecule.

In some embodiments, binding of the anti-TCRβV antibody molecule to aTCRβV region results in one, two, three, four, five, six, seven, eight,nine, ten or more (e.g., all) of the following:

(i) reduced level, e.g., expression level, and/or activity of IL-1β;(ii) reduced level, e.g., expression level, and/or activity of IL-6;(iii) reduced level, e.g., expression level, and/or activity of TNFα;(iv) increased level, e.g., expression level, and/or activity of IL-2;(v) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hoursdelay, in increased level, e.g., expression level, and/or activity ofIL-2;(vi) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours delay,in increased level, e.g., expression level, and/or activity of IFNg;(vii) reduced T cell proliferation kinetics;(viii) reduced cytokine storm, e.g., cytokine release syndrome (CRS)and/or neurotoxicity (NT), e.g., as measured by an assay of Example 3;(ix) cell killing, e.g., target cell killing, e.g. cancer cell killing,e.g., as measured by an assay of Example 4;(x) increased level, e.g., expression level, and/or activity of IL-15;or(xi) increased Natural Killer (NK) cell proliferation, e.g., expansion,compared to an antibody that binds to: a CD3 molecule, e.g., CD3 epsilon(CD3e) molecule; or a TCR alpha (TCRα) molecule.

In some embodiments of any of the compositions disclosed herein, bindingof the anti-TCRβV antibody molecule to a TCRβV region results in areduction of at least 2, 5, 10, 20, 50, 100, or 200 fold, or at least2-200 fold (e.g., 5-150, 10-100, 20-50 fold) in the expression level andor activity of IL-1β as measured by an assay of Example 3.

In some embodiments of any of the compositions disclosed herein, bindingof the anti-TCRβV antibody molecule to a TCRβV region results in areduction of at least 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 fold, or at least 2-1000 fold (e.g., 5-900,10-800, 20-700, 50-600, 100-500, or 200-400 fold) in the expressionlevel and or activity of IL-6 as measured by an assay of Example 3.

In some embodiments of any of the compositions disclosed herein, bindingof the anti-TCRβV antibody molecule to a TCRβV region results in areduction of at least 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, or 2000 fold, or at least 2-2000 fold (e.g.,5-1000, 10-900, 20-800, 50-700, 100-600, 200-500, or 300-400 fold) inthe expression level and or activity of TNFα as measured by an assay ofExample 3.

In some embodiments of any of the compositions disclosed herein, bindingof the anti-TCRβV antibody molecule to a TCRβV region results in anincrease of at least 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, or 2000 fold, or at least 2-2000 fold (e.g.,5-1000, 10-900, 20-800, 50-700, 100-600, 200-500, or 300-400 fold) inthe expression level and or activity of IL-2 as measured by an assay ofExample 3.

In some embodiments of any of the compositions disclosed herein, bindingof the anti-TCRβV antibody molecule to a TCRβV region results in anincrease of at least 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, or 2000 fold, or at least 2-2000 fold (e.g.,5-1000, 10-900, 20-800, 50-700, 100-600, 200-500, or 300-400 fold) inthe expression level and or activity of IL-15 as measured by an assay ofExample 4.

In some embodiments of any of the compositions disclosed herein, bindingof the anti-TCRβV antibody molecule results in proliferation, e.g.,expansion, e.g., at least about 1.1-50 fold expansion (e.g., at leastabout 1.5-40 fold, 2-35 fold, 3-30 fold, 5-25 fold, 8-20 fold, or 10-15fold expansion), of a population of Natural Killer (NK) cells. In someembodiments, the expansion of NK cells is at least about 1.1-30 foldexpansion (e.g., at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, or at least about 1.1-5, 5-10, 10-15, 15-20,20-25, or 25-30 fold expansion). In some embodiments, the expansion ofNK cells is measure by an assay of Example 4. In some embodiments, theexpansion of NK cells by, e.g., binding of, the anti-TCRβV antibodymolecule is compared to expansion of an otherwise similar population notcontacted with the anti-TCRβV antibody molecule.

In some embodiments of any of the compositions disclosed herein, bindingof the anti-TCRβV antibody molecule results in cell killing, e.g.,target cell killing, e.g. cancer cell killing. In some embodiments, thecancer cell is a hematological cancer cell or a solid tumor cell. Insome embodiments, the cancer cell is a multiple myeloma cell. In someembodiments, binding of the anti-TCRβV antibody molecule results in cellkilling in vitro or in vivo. In some embodiments, cell killing ismeasured by an assay of Example 4.

In some embodiments of any of the compositions disclosed herein, bindingof the anti-TCRβV antibody molecule to a TCRβV region results in anincrease or decrease of at least 2, 5, 10, 20, 50, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, or 2000 fold, or at least 2-2000 fold(e.g., 5-1000, 10-900, 20-800, 50-700, 100-600, 200-500, or 300-400fold) of any of the activities described herein compared the activity of16G8 or TM23 murine antibody, or a humanized version thereof asdescribed in U.S. Pat. No. 5,861,155.

In an aspect, provided herein is an antibody molecule which binds, e.g.,specifically binds, to a T cell receptor beta variable chain (TCRβV)region (an anti-TCRβV antibody molecule), wherein the anti-TCRβVantibody molecule:

(i) binds specifically to an epitope on TCRβV, e.g., the same or similarepitope as the epitope recognized by an anti-TCRβV antibody molecule asdescribed herein, e.g., a second anti-TCRβV antibody molecule;(ii) shows the same or similar binding affinity or specificity, or both,as an anti-TCRβV antibody molecule as described herein, e.g., a secondanti-TCRβV antibody molecule;(iii) inhibits, e.g., competitively inhibits, the binding of ananti-TCRβV antibody molecule as described herein, e.g., a secondanti-TCRβV antibody molecule;(iv) binds the same or an overlapping epitope with an anti-TCRβVantibody molecule as described herein, e.g., a second anti-TCRβVantibody molecule; or(v) competes for binding, and/or binds the same epitope, with ananti-TCRβV antibody molecule as described herein, e.g., a secondanti-TCRβV antibody molecule,

In some embodiments, the second anti-TCRβV antibody molecule comprisesan antigen binding domain chosen from Table 1 or Table 2, or a sequencesubstantially identical thereto. In some embodiments, the secondanti-TCRβV antibody molecule comprises an antigen binding domain,comprising:

a heavy chain complementarity determining region 1 (HC CDR1), a heavychain complementarity determining region 2 (HC CDR2) and/or a heavychain complementarity determining region 3 (HC CDR3) of SEQ ID NO: 1 orSEQ ID NO: 9; and/or a light chain complementarity determining region 1(LC CDR1), a light chain complementarity determining region 2 (LC CDR2),and/or a light chain complementarity determining region 3 (LC CDR3) ofSEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments of any of the compositions disclosed herein, bindingof the anti-TCRβV antibody molecule to a TCRβV region results in achange in any (e.g., one, two, three, four or all) of (i)-(v) that isdifferent, e.g., an increase or decrease, of at least 2, 5, 10, 20, 50,100-fold, compared the activity of 16G8 or TM23 murine antibody or ahumanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody molecule binds to a TCRBV family (e.g., genefamily), e.g., a TCRBV gene family comprising subfamilies, e.g., asdescribed herein. In some embodiments, the TCRBV family, e.g., genefamily, comprises: a TCRβ V6 subfamily, a TCRβ V10 subfamily, a TCRβ V12subfamily, a TCRβ V5 subfamily, a TCRβ V7 subfamily, a TCRβ V11subfamily, a TCRβ V14 subfamily, a TCRβ V16 subfamily, a TCRβ V18subfamily, a TCRβ V9 subfamily, a TCRβ V13 subfamily, a TCRβ V4subfamily, a TCRβ V3 subfamily, a TCRβ V2 subfamily, a TCRβ V15 v, aTCRβ V30 subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβV28 subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25subfamily, a TCRβ V29 subfamily, a TCRβ V23 subfamily, a TCRβ V21subfamily, a TCRβ V1 subfamily, a TCRβ V17 subfamily, or a TCRβ V26subfamily.

In some embodiments, the anti-TCRβV antibody binds to a TCRβ V6subfamily chosen from: TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβV6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβV6-3*01 or TCRβ V6-1*01. In some embodiments the TCRβ V6 subfamilycomprises TCRβ V6-5*01.

In some embodiments, the anti-TCRβV antibody binds to a TCRβ V10subfamily chosen from: TCRβ V10-1*01, TCRβ V10-1*02, TCRβ V10-3*01 orTCRβ V10-2*01.

In some embodiments, the anti-TCRβV antibody binds to a TCRβ V12subfamily chosen from: TCRβ V12-4*01, TCRβ V12-3*01 or TCRβ V12-5*01.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody molecule does not bind to TCRβ V12, or binds to TCRβV12 with an affinity and/or binding specificity that is less than (e.g.,less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-,5-, or 10-fold) the affinity and/or binding specificity of the 16G8murine antibody or a humanized version thereof as described in U.S. Pat.No. 5,861,155.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody molecule binds to TCRβ V12 with an affinity and/orbinding specificity that is greater than (e.g., greater than about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) theaffinity and/or binding specificity of the 16G8 murine antibody or ahumanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody molecule binds to a TCRβV region other than TCRβ V12(e.g., TCRβV region as described herein, e.g., TCRβ V6 subfamily (e.g.,TCRβ V6-5*01) with an affinity and/or binding specificity that isgreater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/or bindingspecificity of the 16G8 murine antibody or a humanized version thereofas described in U.S. Pat. No. 5,861,155.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody molecule does not comprise at least one CDR ofAntibody B. In some embodiments of any of the compositions disclosedherein, the anti-TCRβV antibody molecule does not comprise the CDRs ofAntibody B.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody binds to a TCRβ V5 subfamily chosen from: TCRβV5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-8*01, TCRβ V5-1*01.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody binds to a TCRβ V5 subfamily chosen from: TCRβV5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-8*01, TCRβ V5-1*01.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody molecule does not bind to TCRβ V5-5*01 or TCRβV5-1*01, or binds to TCRβ V5-5*01 or TCRβ V5-1*01 with an affinityand/or binding specificity that is less than (e.g., less than about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) theaffinity and/or binding specificity of the TM23 murine antibody or ahumanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody molecule binds to TCRβ V5-5*01 or TCRβ V5-1*01 withan affinity and/or binding specificity that is greater than (e.g.,greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about2-, 5-, or 10-fold) the affinity and/or binding specificity of the TM23murine antibody or a humanized version thereof as described in U.S. Pat.No. 5,861,155.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody molecule binds to a TCRβV region other than TCRβV5-5*01 or TCRβ V5-1*01 (e.g., TCRβV region as described herein, e.g.,TCRβ V6 subfamily (e.g., TCRβ V6-5*01) with an affinity and/or bindingspecificity that is greater than (e.g., greater than about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) theaffinity and/or binding specificity of the TM23 murine antibody or ahumanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody molecule does not comprise at least one CDR of theTM23 murine antibody. In some embodiments of any of the compositionsdisclosed herein, the anti-TCRβV antibody molecule does not comprise theCDRs of the TM23 murine antibody.

In some embodiments of any of the compositions disclosed herein, ananti-TCRβV antibody molecule disclosed herein does not comprise thesequence of a murine anti-rat TCR antibody R73, e.g., as disclosed in JExp Med. 1989 Jan. 1; 169(1): 73-86, herein incorporated by reference inits entirety. In some embodiments of any of the compositions disclosedherein, a multispecific antibody molecule disclosed herein does notcomprise the sequence of a murine anti-rat TCR antibody R73, e.g., asdisclosed in J Immunol. 1993 Mar. 15; 150(6):2305-15, hereinincorporated by reference in its entirety.

In some embodiments of any of the compositions disclosed herein, ananti-TCRβV antibody molecule disclosed herein does not comprise a viralpeptide-WIC complex, e.g., as disclosed in Oncoimmunology. 2016; 5(1):e1052930, herein incorporated by reference in its entirety. In someembodiments of any of the compositions disclosed herein, a multispecificantibody molecule disclosed herein does not comprise a viral peptide-WICcomplex, e.g., as disclosed in Oncoimmunology. 2016; 5(1): e1052930,herein incorporated by reference in its entirety.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody molecule binds to one or more (e.g., all) of thefollowing TCRβV subfamilies:

(i) TCRβ V6 subfamily comprising, e.g., one or more of TCRβ V6-4*01,TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02,TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01;(ii) TCRβ V10 subfamily comprising, e.g., one or more of TCRβ V10-1*01,TCRβ V10-1*02, TCRβ V10-3*01 or TCRβ V10-2*01;(iii) TCRβ V5 subfamily comprising, e.g., one or more of TCRβ V5-6*01,TCRβ V5-4*01, or TCRβ V5-8*01;(iv) TCRβ V12 subfamily comprising e.g., one or more of TCRβ V12-4*01,TCRβ V12-3*01, or TCRβ V12-5*01;(v) TCRβ V7 subfamily comprising e.g., one or more of TCRβ V7-7*01, TCRβV7-6*01, TCRβ V7-8*02, TCRβ V7-4*01, TCRβ V7-2*02, TCRβ V7-2*03, TCRβV7-2*01, TCRβ V7-3*01, TCRβ V7-9*03, or TCRβ V7-9*01;(vi) TCRβ V11 subfamily comprising e.g., one or more of TCRβ V11-1*01,TCRβ V11-2*01 or TCRβ V11-3*01;(vii) TCRβ V14 subfamily comprising TCRβ V14*01;(viii) TCRβ V16 subfamily comprising TCRβ V16*01;(ix) TCRβ V18 subfamily comprising TCRβ V18*01;(x) TCRβ V9 subfamily comprising T e.g., one or more of CRβ V9*01 orTCRβ V9*02;(xi) TCRβ V13 subfamily comprising TCRβ V13*01;(xii) TCRβ V4 subfamily comprising e.g., one or more of e.g., one ormore of TCRβ V4-2*01, TCRβ V4-3*01, or TCRβ V4-1*01;(xiii) TCRβ V3 subfamily comprising TCRβ V3-1*01;(xiv) TCRβ V2 subfamily comprising TCRβ V2*01;(xv) TCRβ V15 subfamily comprising TCRβ V15*01;(xvi) TCRβ V30 subfamily comprising e.g., one or more of TCRβ V30*01, orTCRβ V30*02;(xvii) TCRβ V19 subfamily comprising e.g., one or more of TCRβ V19*01,or TCRβ V19*02;(xviii) TCRβ V27 subfamily comprising TCRβ V27*01;(xix) TCRβ V28 subfamily comprising TCRβ V28*01;(xx) TCRβ V24 subfamily comprising TCRβ V24-1*01;(xxi) TCRβ V20 subfamily comprising e.g., one or more of TCRβ V20-1*01,or TCRβ V20-1*02;(xxii) TCRβ V25 subfamily comprising TCRβ V25-1*01; or(xxiii) TCRβ V29 subfamily comprising TCRβ V29-1*01;(xxiv) TCRβ V21 subfamily;(xxv) TCRβ V1 subfamily;(xxvi) TCRβ V17 subfamily;(xvii) TCRβ V23 subfamily; or(xviii) TCRβ V26 subfamily.

In some embodiments of any of the compositions disclosed herein, theanti-TCRβV antibody molecule binds to one or more (e.g., all) of thefollowing TCRβV subfamilies:

(i) TCRβ V6, e.g., one or more of TCRβ V6-4*01, TCRβ V6-4*02, TCRβV6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβV6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01;(ii) TCRβ V10, e.g., one or more of TCRβ V10-1*01, TCRβ V10-1*02, TCRβV10-3*01 or TCRβ V10-2*01;(iii) TCRβ V12, e.g., one or more of TCRβ V12-4*01, TCRβ V12-3*01, orTCRβ V12-5*01; or(iv) TCRβ V5, e.g., one or more of TCRβ V5-5*01, TCRβ V5-6*01, TCRβV5-4*01, TCR3 V5-8*01, TCRβ V5-1*01.

In some embodiments, the anti-TCRβV antibody molecule binds to TCRβ V6,e.g., one or more of TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβV6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβV6-3*01 or TCRβ V6-1*01. In some embodiments, the anti-TCRβV antibodymolecule binds to TCRβ V6-5*01.

In some embodiments, the anti-TCRβV antibody molecule does not bind toTCRβ V12.

In some embodiments, the anti-TCRβV antibody molecule does not bind toTCRβ V5-5*01 or TCRβ V5-1*01.

In an aspect, provided herein is a multispecific molecule (e.g., abispecific molecule), comprising a first moiety (e.g., a first immunecell engager) comprising an antibody molecule which binds (e.g.,specifically binds) to a T cell receptor beta variable region (TCRβV)(“anti-TCRβV antibody molecule”).

In some embodiments, the multispecific molecule comprises a secondmoiety which comprises one or more of: a tumor-targeting moiety, acytokine molecule, a stromal modifying moiety, or an anti-TCRβV antibodymolecule other than the first moiety.

In some embodiments, binding of the first moiety to the TCRβV regionresults in a cytokine profile, e.g., cytokine secretion profile, thatdiffers from that of a T cell engager that binds to a receptor ormolecule other than a TCRβV region (“a non-TCRβV-binding T cellengager”).

In another aspect, the disclosure provides a multispecific molecule,e.g., a bispecific molecule, comprising the anti-TCRβV antibody moleculedisclosed herein.

In some embodiments, the multispecific molecule further comprises: atumor-targeting moiety, a cytokine molecule, an immune cell engager,e.g., a second immune cell engager, and/or a stromal modifying moiety.

In yet another aspect, disclosed herein is a multispecific molecule,e.g., a bispecific molecule, comprising:

(i) a first moiety comprising a first immune cell engager comprising ananti-TCRβV antibody molecule disclosed herein; and(ii) a second moiety comprising one or more of: a tumor-targetingmoiety; a second immune cell engager; a cytokine molecule or a stromalmodifying moiety.

In another aspect, the disclosure provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding an anti-TCRβVantibody molecule disclosed herein, or a nucleotide sequence having atleast 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

In another aspect, the disclosure provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a multispecificmolecule disclosed herein, or a nucleotide sequence having at least 75%,80%, 85%, 90%, 95%, or 99% identity thereto.

In yet another aspect, the disclosure provides a vector, e.g., anexpression vector, comprising a nucleotide sequence encoding ananti-TCRβV antibody molecule disclosed herein, or a nucleotide sequencehaving at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

In another aspect, the disclosure provides a vector, e.g., an expressionvector, comprising a nucleotide sequence encoding a multispecificmolecule disclosed herein, or a nucleotide sequence having at least 75%,80%, 85%, 90%, 95%, or 99% identity thereto.

In one aspect, the disclosure provides a cell, e.g., host cell, e.g., apopulation of cells, comprising a nucleic acid molecule encoding ananti-TCRβV antibody molecule disclosed herein, or a nucleotide sequencehaving at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. Insome embodiments, the cell or population of cells comprising a nucleicacid molecule encoding anti-TCRβV antibody molecule, comprises: (i) aheavy chain comprising: a variable region (VH), e.g., a VH listed inTable 1 or 2, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or99% identity thereto; and one or more heavy chain constant regions,e.g., as described herein; and/or (ii) a light chain comprising: avariable region (VL) e.g., a VL listed in Table 1 or 2, or a sequencehaving at least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and alight chain constant region, e.g., as described herein, e.g., a kappachain constant region comprising the sequence of SEQ ID NO: 39, or asequence with at least 85%, 90%, 95%, or 99% sequence identity thereto.In some embodiments, the cell or population of cells further comprisesan IgJ heavy chain constant region or a fragment thereof. In someembodiments, the IgJ heavy chain constant region comprises the sequenceof SEQ ID NO: 76 or a sequence with at least 85%, 90%, 95%, or 99%sequence identity thereto. In some embodiments, the IgJ is comprised in,e.g., expressed in, the same cell or population of cells comprising,e.g., expressing, the anti-TCRβV antibody molecule, e.g., the heavychain and/or the light chain of the anti-TCRβV antibody molecule. Insome embodiments, the IgJ is expressed in a different cell or populationof cells than the cell or population of cells comprising, e.g.,expressing, the anti-TCRβV antibody molecule, e.g., the heavy chainand/or the light chain of the anti-TCRβV antibody molecule.

In one aspect, the disclosure provides a cell, e.g., host cell, e.g., apopulation of cells, comprising a nucleic acid molecule encoding amultispecific molecule disclosed herein, or a nucleotide sequence havingat least 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

In one aspect, disclosed herein is an anti-TCRβV antibody molecule foruse in the manufacture of a medicament for treating a disease, e.g.,cancer, in a subject.

In one aspect, disclosed herein is a multispecific molecule comprisingan anti-TCRβV antibody molecule for use in the manufacture of amedicament for treating a disease, e.g., cancer, in a subject.

In another aspect, the disclosure provides a method of making, e.g.,producing, an anti-TCRβV antibody molecule, a multispecific moleculedescribed herein, comprising culturing a host cell described herein,under suitable conditions. In some embodiments of a method of making amultispecific molecule, the conditions comprise, e.g., conditionssuitable for gene expression and/or homo- or heterodimerization.

In another aspect, the disclosure provides a pharmaceutical compositioncomprising an anti-TCRβV antibody molecule, or a multispecific moleculedescribed herein, and a pharmaceutically acceptable carrier, excipient,or stabilizer.

In an aspect, the disclosure provides a method of modulating, e.g.,enhancing, an immune response in a subject comprising administering tothe subject an effective amount of an antibody molecule which binds(e.g., specifically binds) to a T cell receptor beta variable region(TCRβV) (“anti-TCRβV antibody molecule”).

In an aspect, the disclosure provides a method of modulating, e.g.,enhancing, an immune response in a subject comprising administering tothe subject an effective amount of a multispecific molecule disclosedherein.

In some embodiments, the method comprises expanding, e.g., increasingthe number of, an immune cell population in the subject.

In an aspect, the disclosure provides a method of expanding, e.g.,increasing the number of, an immune cell population comprising,contacting the immune cell population with an effective amount of anantibody molecule which binds (e.g., specifically binds) to a T cellreceptor beta variable region (TCRβV) (“anti-TCRβV antibody molecule”).

In an aspect, the disclosure provides a method of expanding, e.g.,increasing the number of, an immune cell population comprising,contacting the immune cell population with an effective amount of amultispecific molecule disclosed herein.

In some embodiments, the expansion occurs in vivo or ex vivo (e.g., invitro).

In some embodiments, the immune cell population comprises a TCRβVexpressing cell, e.g., a TCRβV+cell.

In some embodiments, the TCRβV expressing cell is a T cell, e.g., a CD8+T cell, a CD3+ T cell or a CD4+ T cell.

In some embodiments, the immune cell population comprises a T cell(e.g., a CD4 T cell, a CD8 T cell, (e.g., an effector T cell or a memoryT cell (e.g., a memory effector T cell (e.g., TEM cell, e.g., TEMRAcell), or a tumor infiltrating lymphocyte (TIL).

In some embodiments, the immune cell population comprises a T cell, aNatural Killer cell, a B cell, or a myeloid cell.

In some embodiments, the immune cell population is obtained from ahealthy subject.

In an aspect, provided herein is a method of treating a disease e.g.,cancer, in a subject comprising administering to the subject aneffective amount, e.g., a therapeutically effective amount, of ananti-TCRβV antibody molecule or a multispecific molecule comprising ananti-TCRβV antibody molecule disclosed herein, thereby treating thedisease.

In a related aspect, provided herein is a composition comprising ananti-TCRβV antibody molecule or a multispecific molecule comprising ananti-TCRβV antibody molecule disclosed herein, for use in the treatmentof a disease, e.g., cancer, in a subject.

In some embodiments, the disease is a cancer, e.g., a solid tumor or ahematological cancer, or a metastatic lesion.

In some embodiments, the method further comprises administering a secondagent, e.g., therapeutic agent, e.g., as described herein. In someembodiments, second agent comprises a therapeutic agent (e.g., achemotherapeutic agent, a biologic agent, hormonal therapy), radiation,or surgery. In some embodiments, therapeutic agent is selected from: achemotherapeutic agent, or a biologic agent.

In another aspect, provided herein is a method of targeting, e.g.,directing or re-directing, a therapy, e.g., treatment, to a T cell,e.g., in a subject, e.g., having a disease, e.g., cancer, comprisingadministering an effective amount of: (i) an anti-TCRβV antibodydisclosed herein; and (ii) the therapy, e.g., a tumor targeting therapy(e.g., an antibody that binds to a cancer antigen), e.g., as describedherein, thereby targeting the T cell.

In some embodiments, (i) and (ii) are conjugated, e.g., linked.

In some embodiments, (i) and (ii) are administered simultaneously orconcurrently.

In some embodiments, the method results in: reduced cytokine releasesyndrome (CRS) (e.g., lesser duration of CRS or no CRS), or a reducedseverity of CRS (e.g., absence of severe CRS, e.g., CRS grade 4 or 5)compared to administration of (ii) alone. In some embodiments, CRS isassessed by an assay of Example 3. In some embodiments, the methodresults in: reduced neurotoxicity (NT) (e.g., lesser duration of NT orno NT), or a reduced severity of NT (e.g., absence of severe NT)compared to administration of (ii) alone.

In yet another aspect, the disclosure provides, a method of targeting aT cell, e.g., in a subject having a disease, e.g., cancer, with ananti-TCRβV antibody disclosed herein or a multispecific moleculecomprising an anti-TCRβV antibody disclosed herein.

In another aspect, the disclosure provides a method of treating, e.g.,preventing or reducing, cytokine release syndrome (CRS) and/orneurotoxicity (NT) in a subject, e.g., CRS and/or NT associated with atreatment, e.g., a previously administered treatment, comprisingadministering to the subject an effective amount of an anti-TCRβVantibody disclosed herein or a multispecific molecule comprising ananti-TCRβV antibody disclosed herein, wherein, the subject has adisease, e.g., a cancer, thereby treating, e.g., preventing or reducing,CRS and/or NT in the subject.

In a related aspect, the disclosure provides a composition comprising ananti-TCRβV antibody disclosed herein or a multispecific moleculecomprising an anti-TCRβV antibody disclosed herein, for use in thetreatment, e.g., prevention or reduction, of cytokine release syndrome(CRS) and/or neurotoxicity (NT) in a subject, e.g., CRS and/or NTassociated with a treatment, e.g., a previously administered treatment,comprising administering to the subject an effective amount of theanti-TCRβV antibody, wherein the subject has a disease, e.g., a cancer.

In some embodiments of a method or composition for use disclosed herein,the anti-TCRβV antibody is administered concurrently with or after theadministration of the treatment associated with CRS and/or NT.

In another aspect, provided herein is a method of expanding, e.g.,increasing the number of, an immune cell population comprising,contacting the immune cell population with an antibody molecule, e.g.,humanized antibody molecule, which binds, e.g., specifically binds, to aT cell receptor beta variable chain (TCRβV) region (e.g., anti-TCRβVantibody molecule described herein or a multispecific moleculecomprising an anti-TCRβV antibody molecule described herein), therebyexpanding the immune cell population.

In some embodiments, the expansion occurs in vivo or ex vivo (e.g., invitro).

In an aspect, provided herein is a method of evaluating a subject havinga cancer, comprising acquiring a value of the status of a TCRβV moleculefor the subject, wherein said value comprises a measure of the presenceof, e.g., level or activity of, a TCRβV molecule in a sample from thesubject, wherein the value of the status of a TCRβV molecule is higher,e.g., increased, in a sample from the subject compared to a referencevalue, e.g., a value from a healthy subject, e.g., a subject that doesnot have cancer.

In another aspect, the disclosure provides a method of treating asubject having a cancer, the method comprising (i) acquiring a value ofthe status of a TCRβV molecule for the subject, wherein said valuecomprises a measure of the presence of, e.g., level or activity of, aTCRβV molecule in a sample from the subject, and (ii) responsive to saidvalue, administering an effective amount of an anti-TCRβV antibodymolecule described herein (e.g., a TCRβV agonist) to the subject,thereby treating the cancer.

In some embodiments, the value is higher, e.g., increased, in a samplefrom the subject compared to a reference value, e.g., a value from ahealthy subject, e.g., a subject that does not have cancer.

In a related aspect, the disclosure provides a composition comprising ananti-TCRβV antibody molecule for use in the treatment of a subjecthaving a cancer, comprising (i) acquiring a value of the status of aTCRβV molecule for the subject, wherein said value comprises a measureof the presence of, e.g., level or activity of, a TCRβV molecule in asample from the subject, and (ii) responsive to said value,administering an effective amount of an anti-TCRβV antibody moleculedescribed herein (e.g., a TCRβV agonist) to the subject.

In an aspect, provided herein is method of evaluating a subject for thepresence of a cancer, the method comprising:

(i) acquiring a value of the status of one or more TCRβV molecules forthe subject, e.g., in a biological sample from the subject, wherein saidvalue comprises a measure of the presence of, e.g., level or activityof, a TCRβV molecule in a sample from the subject, and(ii) determining whether the value for the one or more TCRβV moleculesis higher, e.g., increased, in a sample from the subject compared to areference value, e.g., a value from a healthy subject, e.g., a subjectthat does not have cancer, wherein a value that is higher, e.g.,increased, in the subject relative to the reference, e.g., healthysubject, is indicative of the presence of cancer in the subject.

In another aspect, the disclosure provides, a method of treating asubject having cancer, the method comprising:

(i) acquiring a value of the status of one or more TCRβV molecules forthe subject, e.g., in a biological sample from the subject, wherein saidvalue comprises a measure of the presence of, e.g., level or activityof, a TCRβV molecule in a sample from the subject;(ii) determining whether the value for the one or more TCRβV moleculesis higher, e.g., increased, in a sample from the subject compared to areference value, e.g., a value from a healthy subject, e.g., a subjectthat does not have cancer, and(iii) if a value that is higher, e.g., increased, in the subjectrelative to the reference value is determined, administering aneffective amount of an anti-TCRβV antibody molecule, e.g., as describedherein (e.g., a TCRβV agonist), to the subject, thereby treating thecancer.

In a related aspect, provided herein is a composition comprisinganti-TCRβV antibody molecule for use in a method of treating a subjecthaving a cancer, comprising

(i) acquiring a value of the status of one or more TCRβV molecules forthe subject, e.g., in a biological sample from the subject, wherein saidvalue comprises a measure of the presence of, e.g., level or activityof, a TCRβV molecule in a sample from the subject;(ii) determining whether the value for the one or more TCRβV moleculesis higher, e.g., increased, in a sample from the subject compared to areference value, e.g., a value from a healthy subject, e.g., a subjectthat does not have cancer, and(iii) if a value that is higher, e.g., increased, in the subjectrelative to the reference value is determined, administering aneffective amount of an anti-TCRβV antibody molecule, e.g., as describedherein (e.g., a TCRβV agonist), to the subject.

In some embodiments of any of the methods of treatment, or compositionfor use disclosed herein, the status is indicative of the subject havingcancer, or a symptom thereof.

In some embodiments of any of the methods of treatment or compositionfor use disclosed herein, the status is indicative of responsiveness toa therapy, e.g., a therapy comprising an anti-TCRβV antibody molecule,e.g., as described herein.

In some embodiments of any of the methods of treatment or compositionfor use disclosed herein, the value of the status is determined, e.g.,measured, by an assay described herein.

In yet another aspect, provided herein is a method of treating a subjecthaving a cancer, comprising administering to the subject an effectiveamount of an anti-TCRBV antibody molecule described herein, wherein thesubject has a higher, e.g., increased, level or activity of one or moreTCRBV molecules, e.g., as described herein, compared to a referencelevel or activity of one or more TCRBV molecules, e.g., in a healthysubject, e.g., a subject not having a cancer

In an aspect, the disclosure provides, method of treating a subjecthaving a cancer, comprising

(i) isolating a biological sample from the subject; e.g., a peripheralblood sample, biopsy sample, or bone marrow sample; and(ii) acquiring a value of the status of one or more TCRβ V molecules forthe subject, e.g., in the biological sample from the subject, whereinsaid value comprises a measure of the presence of, e.g., level oractivity of, a TCRBV molecule in a sample from the subject compared to areference value, e.g., a sample from a health subject, wherein a valuethat is higher, e.g., increased, in the subject relative to thereference, e.g., healthy subject, is indicative of the presence ofcancer in the subject,(iii) contacting the biological sample with an anti-TCRBV antibodymolecule, e.g., in vitro; and(iv) administering the biological sample or a portion thereof from step(iii) to the subject.

In another aspect, provided herein is method of expanding a populationof immune effector cells from a subject having a cancer, the methodcomprising:

(i) isolating a biological sample comprising a population of immuneeffector cells from the subject; e.g., a peripheral blood sample, biopsysample, or bone marrow sample;(ii) acquiring a value of the status of one or more TCRβ V molecules forthe subject, e.g., in the biological sample from the subject, whereinsaid value comprises a measure of the presence of, e.g., level oractivity of, a TCRBV molecule in a sample from the subject compared to areference value, e.g., a sample from a health subject, wherein a valuethat is higher, e.g., increased, in the subject relative to thereference, e.g., healthy subject, is indicative of the presence ofcancer in the subject, and(iii) contacting the biological sample comprising a population of immuneeffector cells with an anti-TCRβV antibody molecule.

In some embodiments, the method further comprises administering thepopulation of immune effector cells contacted with the anti-TCRβVantibody molecule to the subject.

In some embodiments, a method of expansion, or method of treatment, orcomposition for use disclosed herein comprises measuring T cell function(e.g., cytotoxic activity, cytokine secretion, or degranulation) in thepopulation of immune effector cells, e.g., compared to a referencepopulation, e.g., an otherwise similar population not contacted with theanti-TCRβV antibody molecule or a population of immune effector cellsobtained from a healthy subject (e.g., a subject that does not have acancer).

In some embodiments of any of the methods or composition for usedisclosed herein, the biological sample comprising the population ofimmune effector cells is contacted with an anti-TCRβV antibody moleculethat binds to the one or more TCRβV molecules (e.g., the same TCRβVmolecule) identified as being higher, e.g., increased, in the biologicalsample.

In some embodiments of any of the methods or composition for usedisclosed herein, the biological sample comprising the population ofimmune effector cells is contacted with an anti-TCRβV antibody moleculethat does not bind to the one or more TCRβV molecules (e.g., a differentTCRβV molecule) identified as being higher, e.g., increased, in thebiological sample.

In another aspect, provided herein is a method of identifying one ormore TCRβV molecules associated with a cancer, the method comprising:

(i) acquiring a status for a plurality of TCRβV molecules in abiological sample from a first subject having the disease and in abiological sample from a second subject not having the disease; and(ii) determining whether the level or activity of one or more of theTCRβV molecules is higher, e.g., increased, in the first subjectrelative to the second subject; thereby identifying one or more TCRβVmolecules associated with the cancer.

In some embodiments of any of the methods or composition for usedisclosed herein, the one or more of the TCRβV molecules comprises oneor more, (e.g., all) of the following TCRβV subfamilies:

(i) TCRβ V6 subfamily comprising, e.g., one or more of TCRβ V6-4*01,TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02,TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01;(ii) TCRβ V10 subfamily comprising, e.g., one or more of TCRβ V10-1*01,TCRβ V10-1*02, TCRβ V10-3*01 or TCRβ V10-2*01;(iii) TCRβ V5 subfamily comprising, e.g., one or more of TCRβ V5-6*01,TCRβ V5-4*01, or TCRβ V5-8*01;(iv) TCRβ V12 subfamily comprising e.g., one or more of TCRβ V12-4*01,TCRβ V12-3*01, or TCRβ V12-5*01;(v) TCRβ V7 subfamily comprising e.g., one or more of TCRβ V7-7*01, TCRβV7-6*01, TCRβ V7-8*02, TCRβ V7-4*01, TCRβ V7-2*02, TCRβ V7-2*03, TCRβV7-2*01, TCRβ V7-3*01, TCRβ V7-9*03, or TCRβ V7-9*01;(vi) TCRβ V11 subfamily comprising e.g., one or more of TCRβ V11-1*01,TCRβ V11-2*01 or TCRβ V11-3*01;(vii) TCRβ V14 subfamily comprising TCRβ V14*01;(viii) TCRβ V16 subfamily comprising TCRβ V16*01;(ix) TCRβ V18 subfamily comprising TCRβ V18*01;(x) TCRβ V9 subfamily comprising T e.g., one or more of CRβ V9*01 orTCRβ V9*02;(xi) TCRβ V13 subfamily comprising TCRβ V13*01;(xii) TCRβ V4 subfamily comprising e.g., one or more of e.g., one ormore of TCRβ V4-2*01, TCRβ V4-3*01, or TCRβ V4-1*01;(xiii) TCRβ V3 subfamily comprising TCRβ V3-1*01;(xiv) TCRβ V2 subfamily comprising TCRβ V2*01;(xv) TCRβ V15 subfamily comprising TCRβ V15*01;(xvi) TCRβ V30 subfamily comprising e.g., one or more of TCRβ V30*01, orTCRβ V30*02;(xvii) TCRβ V19 subfamily comprising e.g., one or more of TCRβ V19*01,or TCRβ V19*02;(xviii) TCRβ V27 subfamily comprising TCRβ V27*01;(xix) TCRβ V28 subfamily comprising TCRβ V28*01;(xx) TCRβ V24 subfamily comprising TCRβ V24-1*01;(xxi) TCRβ V20 subfamily comprising e.g., one or more of TCRβ V20-1*01,or TCRβ V20-1*02;(xxii) TCRβ V25 subfamily comprising TCRβ V25-1*01; or(xxiii) TCRβ V29 subfamily comprising TCRβ V29-1*01;(xxiv) TCRβ V21 subfamily;(xxv) TCRβ V1 subfamily;(xxvi) TCRβ V17 subfamily;(xvii) TCRβ V23 subfamily; or(xviii) TCRβ V26 subfamily.

In some embodiments of any of the methods or composition for usedisclosed herein, the cancer is a solid tumor including but not limitedto: melanoma, pancreatic (e.g., pancreatic adenocarcinoma) cancer,breast cancer, colorectal cancer (CRC), lung cancer (e.g., small ornon-small cell lung cancer), skin cancer, ovarian cancer, or livercancer.

In some embodiments of any of the methods or composition for usedisclosed herein, the cancer is a hematological cancer including, butnot limited to: a B-cell or T cell malignancy, e.g., Hodgkin's lymphoma,Non-Hodgkin's lymphoma (e.g., B cell lymphoma, diffuse large B celllymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia(B-CLL), mantle cell lymphoma, marginal zone B-cell lymphoma, Burkittlymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia), acutemyeloid leukemia (AML), chronic myeloid leukemia, myelodysplasticsyndrome, multiple myeloma, and acute lymphocytic leukemia.

In some embodiments of a method of expansion, or method of treatment, orcomposition for use disclosed herein, a higher, e.g., increased, levelor activity of one or more TCRβV molecules in a subject, e.g., in asample from a subject, is indicative of a bias, e.g., a preferentialexpansion, e.g., clonal expansion, of T cells expressing said one ormore TCRβV molecules in the subject.

In some embodiments, a subject having a cancer, e.g., as disclosedherein, has a higher, e.g., increased, level or activity of one or moreTCRβV molecules associated with the cancer. In some embodiments, theTCRβV molecule is associated with, e.g., recognizes, a cancer antigen,e.g., a cancer associated antigen or a neoantigen.

In some embodiments of any of the methods or composition for usedisclosed herein, the subject has B-CLL. In some embodiments, a subjecthaving B-CLL has a higher, e.g., increased, level or activity of one ormore TCRβV molecules, e.g., one or more TCRβV molecules comprising: (i)TCRβ V6 subfamily comprising, e.g., TCRβ V6-4*01, TCRβ V6-4*02, TCRβV6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβV6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01; (ii) TCRβ V5 subfamily comprisingTCRβ V5-6*01, TCRβ V5-4*01, or TCRβ V5-8*01; (iii) TCRβ V3 subfamilycomprising TCRβ V3-1*01; (iv) TCRβ V2 subfamily comprising TCRβ V2*01;or (v) TCRβ V19 subfamily comprising TCRβ V19*01, or TCRβ V19*02.

In some embodiments, a subject having B-CLL has a higher, e.g.,increased, level or activity of a TCRβ V6 subfamily comprising, e.g.,TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01,TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01.In some embodiments, the subject is administered an anti-TCRβV molecule(e.g., an agonistic anti-TCRβV molecule as described herein) that bindsto one or more members of the TCRβ V6 subfamily. In some embodiments,administration of the an anti-TCRβV molecule results in expansion ofimmune cells expressing one or more members of the TCRβ V6 subfamily.

In some embodiments, a subject having B-CLL has a higher, e.g.,increased, level or activity of a TCRβ V5 subfamily comprising TCRβV5-6*01, TCRβ V5-4*01, or TCRβ V5-8*01. In some embodiments, the subjectis administered an anti-TCRβV molecule (e.g., an agonistic anti-TCRβVmolecule as described herein) that binds to one or more members of theTCRβ V5 subfamily. In some embodiments, administration of the ananti-TCRβV molecule results in expansion of immune cells expressing oneor more members of the TCRβ V5 subfamily.

In some embodiments, a subject having B-CLL has a higher, e.g.,increased, level or activity of a TCRβ V3 subfamily comprising TCRβV3-1*01. In some embodiments, the subject is administered an anti-TCRβVmolecule (e.g., an agonistic anti-TCRβV molecule as described herein)that binds to one or more members of the TCRβ V3 subfamily. In someembodiments, administration of the an anti-TCRβV molecule results inexpansion of immune cells expressing one or more members of the TCRβ V3subfamily.

In some embodiments, a subject having B-CLL has a higher, e.g.,increased, level or activity of a TCRβ V2 subfamily comprising TCRβV2*01. In some embodiments, the subject is administered an anti-TCRβVmolecule (e.g., an agonistic anti-TCRβV molecule as described herein)that binds to one or more members of the TCRβ V2 subfamily. In someembodiments, administration of the an anti-TCRβV molecule results inexpansion of immune cells expressing one or more members of the TCRβ V2subfamily.

In some embodiments, a subject having B-CLL has a higher, e.g.,increased, level or activity of a TCRβ V19 subfamily comprising TCRβV19*01, or TCRβ V19*02. In some embodiments, the subject is administeredan anti-TCRβV molecule (e.g., an agonistic anti-TCRBV molecule asdescribed herein) that binds to one or more members of the TCRβ V19subfamily. In some embodiments, administration of the an anti-TCRβVmolecule results in expansion of immune cells expressing one or moremembers of the TCRβ V19 subfamily.

In some embodiments of any of the methods or composition for usedisclosed herein, the subject has melanoma. In some embodiments, asubject having melanoma has a higher, e.g., increased, level or activityof one or more TCRβV molecules, e.g., one or more TCRβV moleculescomprising the TCRβ V6 subfamily comprising, e.g., TCRβ V6-4*01, TCRβV6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβV6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01. In someembodiments, the subject is administered an anti-TCRβV molecule (e.g.,an agonistic anti-TCRβV molecule as described herein) that binds to oneor more members of the TCRβ V6 subfamily. In some embodiments,administration of the an anti-TCRβV molecule results in expansion ofimmune cells expressing one or more members of the TCRβ V6 subfamily.

In some embodiments of any of the methods or composition for usedisclosed herein, the subject has DLBCL. In some embodiments, a subjecthaving melanoma has a higher, e.g., increased, level or activity of oneor more TCRβV molecules, e.g., one or more TCRβV molecules comprising:(i) TCRβ V13 subfamily comprising TCRβ V13*01; (ii) TCRβ V3 subfamilycomprising TCRβ V3-1*01; or (iii) TCRβ V23 subfamily.

In some embodiments, a subject having DLBCL has a higher, e.g.,increased, level or activity of a TCRβ V13 subfamily comprising TCRβV13*01. In some embodiments, the subject is administered an anti-TCRβVmolecule (e.g., an agonistic anti-TCRβV molecule as described herein)that binds to one or more members of the TCRβ V13 subfamily. In someembodiments, administration of the an anti-TCRβV molecule results inexpansion of immune cells expressing one or more members of the TCRβ V13subfamily.

In some embodiments, a subject having DLBCL has a higher, e.g.,increased, level or activity of a TCRβ V3 subfamily comprising TCRβV3-1*01. In some embodiments, the subject is administered an anti-TCRβVmolecule (e.g., an agonistic anti-TCRβV molecule as described herein)that binds to one or more members of the TCRβ V3 subfamily. In someembodiments, administration of the an anti-TCRβV molecule results inexpansion of immune cells expressing one or more members of the TCRβ V3subfamily.

In some embodiments, a subject having DLBCL has a higher, e.g.,increased, level or activity of a TCRβ V23 subfamily. In someembodiments, the subject is administered an anti-TCRβV molecule (e.g.,an agonistic anti-TCRβV molecule as described herein) that binds to oneor more members of the TCRβ V23 subfamily. In some embodiments,administration of the an anti-TCRβV molecule results in expansion ofimmune cells expressing one or more members of the TCRβ V23 subfamily.

In some embodiments of any of the methods or composition for usedisclosed herein, the subject has CRC. In some embodiments, a subjecthaving melanoma has a higher, e.g., increased, level or activity of oneor more TCRβV molecules, e.g., one or more TCRβV molecules comprising:(i) TCRβ V19 subfamily comprising TCRβ V19*01, or TCRβ V19*02; (ii) TCRβV12 subfamily comprising TCRβ V12-4*01, TCRβ V12-3*01, or TCRβ V12-5*01;(iii) TCRβ V16 subfamily comprising TCRβ V16*01; or (iv) TCRβ V21subfamily.

In some embodiments, a subject having CRC has a higher, e.g., increased,level or activity of a TCRβ V19 subfamily comprising TCRβ V19*01, orTCRβ V19*02. In some embodiments, the subject is administered ananti-TCRβV molecule (e.g., an agonistic anti-TCRβV molecule as describedherein) that binds to one or more members of the TCRβ V19 subfamily. Insome embodiments, administration of the an anti-TCRβV molecule resultsin expansion of immune cells expressing one or more members of the TCRβV19 subfamily.

In some embodiments, a subject having CRC has a higher, e.g., increased,level or activity of a TCRβ V12 subfamily comprising TCRβ V12-4*01, TCRβV12-3*01, or TCRβ V12-5*01. In some embodiments, the subject isadministered an anti-TCRβV molecule (e.g., an agonistic anti-TCRβVmolecule as described herein) that binds to one or more members of theTCRβ V12 subfamily. In some embodiments, administration of the ananti-TCRβV molecule results in expansion of immune cells expressing oneor more members of the TCRβ V12 subfamily.

In some embodiments, a subject having CRC has a higher, e.g., increased,level or activity of a TCRβ V16 subfamily comprising TCRβ V16*01. Insome embodiments, the subject is administered an anti-TCRβV molecule(e.g., an agonistic anti-TCRβV molecule as described herein) that bindsto one or more members of the TCRβ V16 subfamily. In some embodiments,administration of the an anti-TCRβV molecule results in expansion ofimmune cells expressing one or more members of the TCRβ V16 subfamily.

In some embodiments, a subject having CRC has a higher, e.g., increased,level or activity of a TCRβ V21 subfamily. In some embodiments, thesubject is administered an anti-TCRβV molecule (e.g., an agonisticanti-TCRβV molecule as described herein) that binds to one or moremembers of the TCRβ V21 subfamily. In some embodiments, administrationof the an anti-TCRβV molecule results in expansion of immune cellsexpressing one or more members of the TCRβ V21 subfamily.

Alternatively or in combination with any of the embodiments disclosedherein, provided herein is an anti-TCRβV antibody molecule which:

(i) binds specifically to an epitope on TCRβV, e.g., the same or similarepitope as the epitope recognized by an anti-TCRβV antibody molecule asdescribed herein, e.g., a second anti-TCRβV antibody molecule;(ii) shows the same or similar binding affinity or specificity, or both,as an anti-TCRβV antibody molecule as described herein, e.g., a secondanti-TCRβV antibody molecule;(iii) inhibits, e.g., competitively inhibits, the binding of ananti-TCRβV antibody molecule as described herein, e.g., a secondanti-TCRβV antibody molecule;(iv) binds the same or an overlapping epitope with an anti-TCRβVantibody molecule as described herein, e.g., a second anti-TCRβVantibody molecule; or(v) competes for binding, and/or binds the same epitope, with ananti-TCRβV antibody molecule as described herein, e.g., a secondanti-TCRβV antibody molecule,

In some embodiments, the second anti-TCRβV antibody molecule comprisesan antigen binding domain chosen from Table 1 or Table 2, or a sequencesubstantially identical thereto. In some embodiments, the secondanti-TCRβV antibody molecule comprises an antigen binding domain,comprising:

-   -   a heavy chain complementarity determining region 1 (HC CDR1), a        heavy chain complementarity determining region 2 (HC CDR2)        and/or a heavy chain complementarity determining region 3 (HC        CDR3) of SEQ ID NO: 1 or SEQ ID NO: 9; and/or a light chain        complementarity determining region 1 (LC CDR1), a light chain        complementarity determining region 2 (LC CDR2), and/or a light        chain complementarity determining region 3 (LC CDR3) of SEQ ID        NO: 2, SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising:

(i) a heavy chain complementarity determining region 1 (HC CDR1), aheavy chain complementarity determining region 2 (HC CDR2) and/or aheavy chain complementarity determining region 3 (HC CDR3) of SEQ ID NO:1 or SEQ ID NO: 9, or a sequence disclosed in Table 1; or(ii) a light chain complementarity determining region 1 (LC CDR1), alight chain complementarity determining region 2 (LC CDR2), and/or alight chain complementarity determining region 3 (LC CDR3) of SEQ ID NO:2, SEQ ID NO: 10 or SEQ ID NO: 11, or a sequence disclosed in Table 1.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising a light chain variable region (VL) comprising one, twoor all (e.g., three) of a LC CDR1, a LC CDR2 and a LC CDR3 of SEQ ID NO:2, SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising a heavy chain variable region (VH) comprising one, twoor all (e.g., three) of a HC CDR1, a HC CDR2 and a HC CDR3 of SEQ IDNO:1 or SEQ ID NO: 9.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising:

(i) a VL comprising: a LC CDR1 amino acid sequence of SEQ ID NO: 6 (oran amino acid sequence with not more than 1, 2, 3 or 4 modifications,e.g., substitutions, additions or deletions thereof), a LC CDR2 aminoacid sequence of SEQ ID NO:7 (or an amino acid sequence with not morethan 1, 2, 3 or 4 modifications, e.g., substitutions, additions ordeletions thereof), and/or a LC CDR3 amino acid sequence of SEQ ID NO:8(or an amino acid sequence with not more than 1, 2, 3 or 4modifications, e.g., substitutions, additions or deletions thereof);and/or(ii) a VH comprising: a HC CDR1 amino acid sequence of SEQ ID NO: 3 (oran amino acid sequence with not more than 1, 2, 3 or 4 modifications,e.g., substitutions, additions or deletions thereof), a HC CDR2 aminoacid sequence of SEQ ID NO:4 (or an amino acid sequence with not morethan 1, 2, 3 or 4 modifications, e.g., substitutions, additions ordeletions thereof), and/or a HC CDR3 amino acid sequence of SEQ ID NO:5(or an amino acid sequence with not more than 1, 2, 3 or 4modifications, e.g., substitutions, additions or deletions thereof).

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising:

a variable heavy chain (VH) of SEQ ID NO: 9, or a sequence having atleast about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto;and/or a variable light chain (VL) of SEQ ID NO: 10 or SEQ ID NO: 11, ora sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99%sequence identity thereto.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising the VH amino acid sequence of SEQ ID NO: 9 and the VLamino acid sequence of SEQ ID NO: 10.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising the VH amino acid sequence of SEQ ID NO: 9 and the VLamino acid sequence of SEQ ID NO: 11.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a heavy chaincomprising a framework region, e.g., framework region 3 (FR3),comprising one or both of: (i) a Threonine at position 73, e.g., asubstitution at position 73 according to Kabat numbering, e.g., aGlutamic Acid to Threonine substitution; or (ii) a Glycine at position,e.g., a substitution at position 94 according to Kabat numbering, e.g.,a Arginine to Glycine substitution. In some embodiments, thesubstitution is relative to a human germline heavy chain frameworkregion sequence.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a light chaincomprising a framework region, e.g., framework region 1 (FR1),comprising a Phenylalanine at position 10, e.g., a substitution atposition 10 according to Kabat numbering, e.g., a Serine to Phenyalaninesubstitution. In some embodiments, the substitution is relative to ahuman germline light chain framework region sequence.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a light chaincomprising a framework region, e.g., framework region 2 (FR2),comprising one or both of: (i) a Histidine at position 36, e.g., asubstitution at position 36 according to Kabat numbering, e.g., aTyrosine to Histidine substitution; or (ii) an Alanine at position 46,e.g., a substitution at position 46 according to Kabat numbering, e.g.,a Arginine to Alanine substitution. In some embodiments, thesubstitution is relative to a human germline light chain frameworkregion sequence.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a light chaincomprising a framework region, e.g., framework region 3 (FR3),comprising a Phenylalanine at position 87, e.g., a substitution atposition 87 according to Kabat numbering, e.g., a Tyrosine toPhenyalanine substitution. In some embodiments, the substitution isrelative to a human germline light chain framework region sequence.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule binds to TCRβ V6, e.g., TCRβV6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβV6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01. Insome embodiments the anti-TCRβV antibody molecule binds to TCRβ V6-5*01.

In some embodiments, TCRβ V6, e.g., TCRβ V6-4*01, TCRβ V6-4*02, TCRβV6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβV6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01, is recognized, e.g., bound, bySEQ ID NO: 1 and/or SEQ ID NO: 2. In some embodiments, TCRβ V6, e.g.,TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01,TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01,is recognized, e.g., bound, by SEQ ID NO: 9 and/or SEQ ID NO: 10. Insome embodiments, TCRβ V6, e.g., TCRβ V6-4*01, TCRβ V6-4*02, TCRβV6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβV6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01, is recognized, e.g., bound, bySEQ ID NO: 9 and/or SEQ ID NO: 11. In some embodiments, TCRβ V6-5*01 isrecognized, e.g., bound by SEQ ID NO: 9 and/or SEQ ID NO: 10, or asequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequenceidentity thereto. In some embodiments, TCRβ V6-5*01 is recognized, e.g.,bound by SEQ ID NO: 9 and/or SEQ ID NO: 11, or a sequence having atleast about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising:

(i) a heavy chain complementarity determining region (HC CDR1), a HCCDR2 and/or a HC CDR3 of SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 24 orSEQ ID NO: 25, or a sequence disclosed in Table 2; and/or(ii) a light chain complementarity determining region 1 (LC CDR1), a LCCDR2, and/or a LC CDR3 of SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO:30, or a sequence disclosed inTable 2.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising a light chain variable region (VL) comprising one, twoor all of a LC CDR1, a LC CDR2 and a LC CDR3 of SEQ ID NO: 16, SEQ IDNO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO:30.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising a heavy chain variable region (VH) comprising one, twoor all of a HC CDR1, a HC CDR2 and a HC CDR3 of SEQ ID NO: 15, SEQ IDNO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising:

(i) a VL comprising: a LC CDR1 amino acid sequence of SEQ ID NO: 20 (oran amino acid sequence with not more than 1, 2, 3 or 4 modifications,e.g., substitutions, additions or deletions thereof), a LC CDR2 aminoacid sequence of SEQ ID NO:21 (or an amino acid sequence with not morethan 1, 2, 3 or 4 modifications, e.g., substitutions, additions ordeletions thereof), and/or a LC CDR3 amino acid sequence of SEQ ID NO:22(or an amino acid sequence with not more than 1, 2, 3 or 4modifications, e.g., substitutions, additions or deletions thereof);and/or(ii) a VH comprising: a HC CDR1 amino acid sequence of SEQ ID NO: 17 (oran amino acid sequence with not more than 1, 2, 3 or 4 modifications,e.g., substitutions, additions or deletions thereof), a HC CDR2 aminoacid sequence of SEQ ID NO:18 (or an amino acid sequence with not morethan 1, 2, 3 or 4 modifications, e.g., substitutions, additions ordeletions thereof), and/or a HC CDR3 amino acid sequence of SEQ ID NO:19(or an amino acid sequence with not more than 1, 2, 3 or 4modifications, e.g., substitutions, additions or deletions thereof).

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises an antigen bindingdomain comprising:

a variable heavy chain (VH) of SEQ ID NO: 23, SEQ ID NO: 24 or SEQ IDNO: 25, or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or99% sequence identity thereto; and/ora variable light chain (VL) of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:28, SEQ ID NO: 29 or SEQ ID NO:30, or a sequence having at least about75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a light chaincomprising a framework region, e.g., framework region 1 (FR1),comprising one, two or all (e.g., three) of: (i) an Aspartic Acid atposition 1, e.g., a substitution at position 1 according to Kabatnumbering, e.g., a Alanine to Aspartic Acid substitution; or (ii) anAsparagine at position 2, e.g., a substitution at position 2 accordingto Kabat numbering, e.g., a Isoleucine to Asparagine substitution, aSerine to Asparagine substitution, or a Tyrosine to Asparaginesubstitution; or (iii) a Leucine at position 4, e.g., a substitution atposition 4 according to Kabat numbering, e.g., a Methionine to Leucinesubstitution. In some embodiments, the substitution is relative to ahuman germline light chain framework region sequence.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a light chaincomprising a framework region, e.g., framework region 3 (FR3),comprising one, two or all (e.g., three) of: (i) a Glycine as position66, e.g., a substitution at position 66 according to Kabat numbering,e.g., a Lysine to Glycine substitution, or a Serine to Glycinesubstitution; or (ii) an Asparagine at position 69, e.g., a substitutionat position 69 according to Kabat numbering, e.g., a Threonine toAsparagine substitution; or (iii) a Tyrosine at position 71, e.g., asubstitution at position 71 according to Kabat numbering, e.g., aPhenylalanine to Tyrosine substitution, or an Alanine to Tyrosinesubstitution. In some embodiments, the substitution is relative to ahuman germline light chain framework region sequence.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule binds to TCRβ V12, e.g., TCRβV12-4*01, TCRβ V12-3*01, or TCRβ V12-5*01. In some embodiments theanti-TCRβV antibody molecule binds to TCRβ V12-4*01 or TCRβ V12-3*01.

In some embodiments, TCRβ V12, e.g., TCRβ V12-4*01, TCRβ V12-3*01, orTCRβ V12-5*01 is recognized, e.g., bound, by SEQ ID NO: 15 and/or SEQ IDNO: 16. In some embodiments, TCRβ V12, e.g., TCRβ V12-4*01, TCRβV12-3*01, or TCRβ V12-5*01, is recognized, e.g., bound, by any one ofSEQ ID NOs 23-25, and/or any one of SEQ ID NO: 26-30, or an amino acidsequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequenceidentity thereto. In some embodiments TCRβ V12-4*01 is recognized, e.g.,bound, by any one of SEQ ID NOs 23-25, and/or any one of SEQ ID NO:26-30, or an amino acid sequence having at least about 75%, 80%, 85%,90%, 95%, or 99% sequence identity thereto. In some embodiments TCRβV12-3*01 is recognized, e.g., bound, by any one of SEQ ID NOs 23-25,and/or any one of SEQ ID NO: 26-30, or an amino acid sequence having atleast about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises the anti-TCRβVantibody molecule comprises an antigen binding domain comprising asingle chain Fv (scFv) or a Fab.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises binds to aconformational or a linear epitope on the T cell.

In some embodiments of any of the compositions or methods disclosedherein, the tumor comprises an antigen, e.g., a tumor antigen, e.g., atumor associated antigen or a neoantigen. In some embodiments, theanti-TCRβV antibody molecule recognize, e.g., bind to, the tumorantigen.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule is a full antibody (e.g., anantibody that includes at least one, and preferably two, complete heavychains, and at least one, and preferably two, complete light chains), oran antigen-binding fragment (e.g., a Fab, F(ab′)₂, Fv, a single chain Fvfragment, a single domain antibody, a diabody (dAb), a bivalentantibody, or bispecific antibody or fragment thereof, a single domainvariant thereof, a camelid antibody, or a rat-derived VH.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises the anti-TCRβVantibody molecule comprises one or more heavy chain constant regionschosen from IgG1, IgG2, IgG3, IgGA1, IgGA2, IgM, IgJ or IgG4, or afragment thereof, e.g., as disclosed in Table 3.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a heavy chainconstant region of an IgM or a fragment thereof, optionally wherein theIgM heavy chain constant region comprises the sequence of SEQ ID NO: 73,or a sequence with at least 85%, 90%, 95%, or 99% sequence identitythereto. In some embodiments of any of the compositions or methodsdisclosed herein, the anti-TCRβV antibody molecule comprising an IgMconstant region, further comprises a heavy chain constant region of anIgJ or a fragment thereof, optionally wherein the IgJ heavy chainconstant region comprises the sequence of SEQ ID NO: 76 or a sequencewith at least 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a heavy chainconstant region of an IgJ or a fragment thereof, optionally wherein theIgJ heavy chain constant region comprises the sequence of SEQ ID NO: 76or a sequence with at least 85%, 90%, 95%, or 99% sequence identitythereto.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a heavy chainconstant region of an IgGA1, or a fragment thereof, optionally whereinthe IgGA1 heavy chain constant region comprises the sequence of SEQ IDNO: 74, or a sequence with at least 85%, 90%, 95%, or 99% sequenceidentity thereto.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a heavy chainconstant region of an IgGA2, or a fragment thereof, optionally whereinthe IgGA2 heavy chain constant region comprises a sequence listed inTable 3, e.g., SEQ ID NO: 75, or a sequence with at least 85%, 90%, 95%,or 99% sequence identity thereto.

In some embodiments of any of the compositions or methods disclosedherein, binding of the anti-TCRβV antibody molecule to a TCRβV regionresults in a cytokine profile, e.g., a cytokine secretion profile,(e.g., comprising one or more cytokines and/or one or more chemokines),that differs from that of a T cell engager that binds to a receptor ormolecule other than a TCRβV region (“a non-TCRβV-binding T cellengager”).

In some embodiments, the cytokine profile, e.g., cytokine secretionprofile, comprises the level and/or activity of one or more cytokinesand/or one or more chemokines (e.g., as described herein). In anembodiment, a cytokine profile, e.g., a cytokine secretion profile,comprises the level and/or activity of one or more of: IL-2 (e.g., fulllength, a variant, or a fragment thereof); IL-1beta (e.g., full length,a variant, or a fragment thereof); IL-6 (e.g., full length, a variant,or a fragment thereof); TNFα (e.g., full length, a variant, or afragment thereof); IFNg (e.g., full length, a variant, or a fragmentthereof) IL-10 (e.g., full length, a variant, or a fragment thereof);IL-4 (e.g., full length, a variant, or a fragment thereof); TNF alpha(e.g., full length, a variant, or a fragment thereof); IL-12p70 (e.g.,full length, a variant, or a fragment thereof); IL-13 (e.g., fulllength, a variant, or a fragment thereof); IL-8 (e.g., full length, avariant, or a fragment thereof); Eotaxin (e.g., full length, a variant,or a fragment thereof); Eotaxin-3 (e.g., full length, a variant, or afragment thereof); IL-8 (HA) (e.g., full length, a variant, or afragment thereof); IP-10 (e.g., full length, a variant, or a fragmentthereof); MCP-1 (e.g., full length, a variant, or a fragment thereof);MCP-4 (e.g., full length, a variant, or a fragment thereof); MDC (e.g.,full length, a variant, or a fragment thereof); MIP-1a (e.g., fulllength, a variant, or a fragment thereof); MIP-1b (e.g., full length, avariant, or a fragment thereof); TARC (e.g., full length, a variant, ora fragment thereof); GM-CSF (e.g., full length, a variant, or a fragmentthereof); IL-12 23p40 (e.g., full length, a variant, or a fragmentthereof); IL-15 (e.g., full length, a variant, or a fragment thereof);IL-16 (e.g., full length, a variant, or a fragment thereof); IL-17a(e.g., full length, a variant, or a fragment thereof); IL-1a (e.g., fulllength, a variant, or a fragment thereof); IL-5 (e.g., full length, avariant, or a fragment thereof); IL-7 (e.g., full length, a variant, ora fragment thereof); TNF-beta (e.g., full length, a variant, or afragment thereof); or VEGF (e.g., full length, a variant, or a fragmentthereof).

In some embodiments, the cytokine profile, e.g., cytokine secretionprofile, comprises one, two, three, four, five, six, seven, or all ofthe following:

(i) increased level, e.g., expression level, and/or activity of IL-2;(ii) reduced level, e.g., expression level, and/or activity of IL-1β;(iii) reduced level, e.g., expression level, and/or activity of IL-6;(iv) reduced level, e.g., expression level, and/or activity of TNFα;(v) reduced level, e.g., expression level, and/or activity of IL-10;(vi) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hoursdelay, in increased level, e.g., expression level, and/or activity ofIL-2;(vii) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours delay,in increased level, e.g., expression level, and/or activity of IFNg; or(viii) increased level, e.g., expression level, and/or activity ofIL-15, e.g., wherein (i)-(viii) are relative to the cytokine profile,e.g., cytokine secretion profile, of the non-TCRBV-binding T cellengager.

In some embodiments, binding of the anti-TCRBV antibody to a TCRβVregion results in reduced cytokine storm, e.g., reduced cytokine releasesyndrome (CRS) and/or neurotoxicity (NT), as measured by an assay ofExample 3, e.g., relative to the cytokine storm induced by thenon-TCRBV-binding T cell engager.

In some embodiments, binding of the anti-TCRBV antibody to a TCRβVregion results in one, two, three or all of:

(ix) reduced T cell proliferation kinetics;(x) cell killing, e.g., target cell killing, e.g. cancer cell killing,e.g., as measured by an assay of Example 4;(xi) increased Natural Killer (NK) cell proliferation, e.g., expansion;or(xii) expansion, e.g., at least about 1.1-10 fold expansion (e.g., atleast about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 foldexpansion), of a population of memory T cells, e.g., wherein (ix)-(xii)are relative to the non-TCRBV-binding T cell engager.

In some embodiments, an anti-TCRβV antibody molecule disclosed hereinrecognizes (e.g., binds to), a structurally conserved domain on theTCRβV protein (e.g., as denoted by the circled area in FIG. 24A).

In some embodiments, an anti-TCRβV antibody molecule disclosed hereindoes not recognize, e.g., bind to, an interface of a TCRβV:TCRalphacomplex.

In some embodiments, an anti-TCRβV antibody molecule disclosed hereindoes not recognize, e.g., bind to, a constant region of a TCRβV protein.An exemplary antibody that binds to a constant region of a TCRBV regionis JOVI.1 as described in Viney et al., (Hybridoma. 1992 December;11(6):701-13).

In some embodiments, an anti-TCRβV antibody molecule disclosed hereindoes not recognize, e.g., bind to, one or more (e.g., all) of acomplementarity determining region (e.g., CDR1, CDR2 and/or CDR3) of aTCRβV protein.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a light chainconstant region chosen from the light chain constant regions of kappa orlambda, or a fragment thereof, e.g., as disclosed in Table 3.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises a light chainconstant region of a kappa chain, or a fragment thereof, optionallywherein the kappa chain constant region comprises the sequence of SEQ IDNO: 39, or a sequence with at least 85%, 90%, 95%, or 99% sequenceidentity thereto

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises:

(i) one or more heavy chain constant regions comprising a heavy chainconstant region chosen from IgG1, IgG2, IgG3, IgGA1, IgGA2, IgG4, IgJ,IgM, IgD, or IgE, or a fragment thereof, e.g., as described in Table 3;and(ii) a light chain constant region comprising a light chain constantregion chosen from the light chain constant regions of kappa or lambda,or a fragment thereof, e.g., as described in Table 3.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises or a cell comprisingan anti-TCRβV antibody molecule comprises:

(i) a heavy chain comprising a variable region (VH), e.g., a VH of anantibody disclosed herein; and/or one or more heavy chain constantregions, e.g., as disclosed herein; and/or(ii) a light chain comprising a variable light (VL), e.g., a VL of anantibody disclosed herein; and/or one or more light chain constantregions, e.g., as disclosed herein.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises, or a cell comprisingan anti-TCRβV antibody molecule comprises:

(i) a heavy chain comprising a heavy chain constant region comprising:

(a) an IgM heavy chain constant region or a fragment thereof, comprisingthe sequence of SEQ ID NO: 73, or a sequence with at least 85%, 90%,95%, or 99% sequence identity thereto;

(b) an IgGA1 heavy chain constant region or a fragment thereof,comprising the sequence of SEQ ID NO: 74, or a sequence with at least85%, 90%, 95%, or 99% sequence identity thereto; or

(c) an IgGA2 heavy chain constant region or a fragment thereof,comprising the sequence of SEQ ID NO: 75, or a sequence with at least85%, 90%, 95%, or 99% sequence identity thereto; and

(ii) a light chain comprising a light chain constant region comprising akappa chain constant region comprising the sequence of SEQ ID NO: 39, ora sequence with at least 85%, 90%, 95%, or 99% sequence identitythereto,optionally wherein, the anti-TCRβV antibody molecule further comprisesan IgJ heavy chain constant region or a fragment thereof, wherein theIgJ heavy chain constant region comprises the sequence of SEQ ID NO: 76or a sequence with at least 85%, 90%, 95%, or 99% sequence identitythereto.

In some embodiments of any of the compositions or methods disclosedherein, the anti-TCRβV antibody molecule comprises, or a cell comprisingan anti-TCRβV antibody molecule comprises:

(i) a heavy chain comprising: a VH chosen from a VH of Table 1 or 2, ora sequence with at least 85%, 90%, 95%, or 99% sequence identitythereto; and a heavy chain constant region comprising:

(a) an IgM heavy chain constant region or a fragment thereof, comprisingthe sequence of SEQ ID NO: 73, or a sequence with at least 85%, 90%,95%, or 99% sequence identity thereto;

(b) an IgGA1 heavy chain constant region or a fragment thereof,comprising the sequence of SEQ ID NO: 74, or a sequence with at least85%, 90%, 95%, or 99% sequence identity thereto; or

(c) an IgGA2 heavy chain constant region or a fragment thereof,comprising the sequence of SEQ ID NO: 75, or a sequence with at least85%, 90%, 95%, or 99% sequence identity thereto; and

(ii) a light chain comprising: a VL chosen from a VL of Table 1 or 2, ora sequence with at least 85%, 90%, 95%, or 99% sequence identitythereto; and a light chain constant region comprising a kappa chainconstant region comprising the sequence of SEQ ID NO: 39, or a sequencewith at least 85%, 90%, 95%, or 99% sequence identity thereto,optionally wherein, the anti-TCRβV antibody molecule further comprisesan IgJ heavy chain constant region or a fragment thereof, wherein theIgJ heavy chain constant region comprises the sequence of SEQ ID NO: 76or a sequence with at least 85%, 90%, 95%, or 99% sequence identitythereto.

In some embodiments of any of the methods disclosed herein, theanti-TCRβV antibody molecule binds to one or more (e.g., all) of thefollowing TCRβV subfamilies:

(i) TCRβ V6 subfamily comprising, e.g., one or more of TCRβ V6-4*01,TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02,TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01;(ii) TCRβ V10 subfamily comprising, e.g., one or more of TCRβ V10-1*01,TCRβ V10-1*02, TCRβ V10-3*01 or TCRβ V10-2*01;(iii) TCRβ V5 subfamily comprising, e.g., one or more of TCRβ V5-6*01,TCRβ V5-4*01, or TCRβ V5-8*01;(iv) TCRβ V12 subfamily comprising e.g., one or more of TCRβ V12-4*01,TCRβ V12-3*01, or TCRβ V12-5*01;(v) TCRβ V7 subfamily comprising e.g., one or more of TCRβ V7-7*01, TCRβV7-6*01, TCRβ V7-8*02, TCRβ V7-4*01, TCRβ V7-2*02, TCRβ V7-2*03, TCRβV7-2*01, TCRβ V7-3*01, TCRβ V7-9*03, or TCRβ V7-9*01;(vi) TCRβ V11 subfamily comprising e.g., one or more of TCRβ V11-1*01,TCRβ V11-2*01 or TCRβ V11-3*01;(vii) TCRβ V14 subfamily comprising TCRβ V14*01;(viii) TCRβ V16 subfamily comprising TCRβ V16*01;(ix) TCRβ V18 subfamily comprising TCRβ V18*01;(x) TCRβ V9 subfamily comprising T e.g., one or more of CRβ V9*01 orTCRβ V9*02;(xi) TCRβ V13 subfamily comprising TCRβ V13*01;(xii) TCRβ V4 subfamily comprising e.g., one or more of e.g., one ormore of TCRβ V4-2*01, TCRβ V4-3*01, or TCRβ V4-1*01;(xiii) TCRβ V3 subfamily comprising TCRβ V3-1*01;(xiv) TCRβ V2 subfamily comprising TCRβ V2*01;(xv) TCRβ V15 subfamily comprising TCRβ V15*01;(xvi) TCRβ V30 subfamily comprising e.g., one or more of TCRβ V30*01, orTCRβ V30*02;(xvii) TCRβ V19 subfamily comprising e.g., one or more of TCRβ V19*01,or TCRβ V19*02;(xviii) TCRβ V27 subfamily comprising TCRβ V27*01;(xix) TCRβ V28 subfamily comprising TCRβ V28*01;(xx) TCRβ V24 subfamily comprising TCRβ V24-1*01;(xxi) TCRβ V20 subfamily comprising e.g., one or more of TCRβ V20-1*01,or TCRβ V20-1*02;(xxii) TCRβ V25 subfamily comprising TCRβ V25-1*01; or(xxiii) TCRβ V29 subfamily comprising TCRβ V29-1*01;(xxiv) TCRβ V21 subfamily;(xxv) TCRβ V1 subfamily;(xxvi) TCRβ V17 subfamily;(xvii) TCRβ V23 subfamily; or(xviii) TCRβ V26 subfamily.

In some embodiments of any of the methods disclosed herein, theanti-TCRβV antibody molecule binds to one or more (e.g., all) of thefollowing TCRβV subfamilies:

(i) TCRβ V6, e.g., TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβV6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβV6-3*01 or TCRβ V6-1*01; (ii) TCRβ V10, e.g., TCRβ V10-1*01, TCRβV10-1*02, TCRβ V10-3*01 or TCRβ V10-2*01;

(iii) TCRβ V12, e.g., TCRβ V12-4*01, TCRβ V12-3*01, or TCRβ V12-5*01; or

(iv) TCRβ V5, e.g., TCRβ V5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβV5-8*01, TCRβ V5-1*01.

In some embodiments, the anti-TCRβV antibody molecule binds to TCRβ V6,e.g., TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβV6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβV6-1*01. In some embodiments, the anti-TCRβV antibody molecule binds toTCRβ V6-5*01.

In some embodiments, the anti-TCRβV antibody molecule does not bind toTCRβ V12.

In some embodiments, the anti-TCRβV antibody molecule does not bind toTCRβ V5-5*01 or TCRβ V5-1*01.

In some embodiments of any of the methods disclosed herein, theanti-TCRβV antibody molecule does not bind to TCRβ V12, or binds to TCRβV12 with an affinity and/or binding specificity that is less than (e.g.,less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-,5-, or 10-fold) the affinity and/or binding specificity of the 16G8murine antibody or a humanized version thereof as described in U.S. Pat.No. 5,861,155.

In some embodiments of any of the methods disclosed herein, theanti-TCRβV antibody molecule binds to TCRβ V12 with an affinity and/orbinding specificity that is greater than (e.g., greater than about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) theaffinity and/or binding specificity of the 16G8 murine antibody or ahumanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments of any of the methods disclosed herein, theanti-TCRβV antibody molecule binds to a TCRβV region other than TCRβ V12(e.g., TCRβV region as described herein, e.g., TCRβ V6 subfamily (e.g.,TCRβ V6-5*01) with an affinity and/or binding specificity that isgreater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/or bindingspecificity of the 16G8 murine antibody or a humanized version thereofas described in U.S. Pat. No. 5,861,155.

In some embodiments of any of the methods disclosed herein, theanti-TCRβV antibody molecule does not comprise at least one CDR ofAntibody B. In some embodiments of any of the methods disclosed herein,the anti-TCRβV antibody molecule does not comprise the CDRs of AntibodyB.

In some embodiments of any of the methods disclosed herein, theanti-TCRβV antibody molecule does not bind to TCRβ V5-5*01 or TCRβV5-1*01, or binds to TCRβ V5-5*01 or TCRβ V5-1*01 with an affinityand/or binding specificity that is less than (e.g., less than about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) theaffinity and/or binding specificity of the TM23 murine antibody or ahumanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments of any of the methods disclosed herein, theanti-TCRβV antibody molecule binds to TCRβ V5-5*01 or TCRβ V5-1*01 withan affinity and/or binding specificity that is greater than (e.g.,greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about2-, 5-, or 10-fold) the affinity and/or binding specificity of the TM23murine antibody or a humanized version thereof as described in U.S. Pat.No. 5,861,155.

In some embodiments of any of the methods disclosed herein, theanti-TCRβV antibody molecule binds to a TCRβV region other than TCRβV5-5*01 or TCRβ V5-1*01 (e.g., TCRβV region as described herein, e.g.,TCRβ V6 subfamily (e.g., TCRβ V6-5*01) with an affinity and/or bindingspecificity that is greater than (e.g., greater than about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) theaffinity and/or binding specificity of the TM23 murine antibody or ahumanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments of any of the methods disclosed herein, theanti-TCRβV antibody molecule does not comprise at least one CDR of theTM23 murine antibody. In some embodiments of any of the methodsdisclosed herein, the anti-TCRβV antibody molecule does not comprise theCDRs of the TM23 murine antibody.

In some embodiments of any of the methods disclosed herein, ananti-TCRβV antibody molecule disclosed herein does not comprise thesequence of a murine anti-rat TCR antibody R73, e.g., as disclosed in JExp Med. 1989 Jan. 1; 169(1): 73-86, herein incorporated by reference inits entirety. In some embodiments of any of the methods disclosedherein, a multispecific antibody molecule disclosed herein does notcomprise the sequence of a murine anti-rat TCR antibody R73, e.g., asdisclosed in J Immunol. 1993 Mar. 15; 150(6):2305-15, hereinincorporated by reference in its entirety.

In some embodiments of any of the methods disclosed herein, ananti-TCRβV antibody molecule disclosed herein does not comprise a viralpeptide-MHC complex, e.g., as disclosed in Oncoimmunology. 2016; 5(1):e1052930, herein incorporated by reference in its entirety. In someembodiments of any of the methods disclosed herein, a multispecificantibody molecule disclosed herein does not comprise a viral peptide-MHCcomplex, e.g., as disclosed in Oncoimmunology. 2016; 5(1): e1052930,herein incorporated by reference in its entirety.

In some embodiments of a method disclosed herein, the immune cellpopulation comprises a T cell, a Natural Killer cell, a B cell, anantigen presenting cell, or a myeloid cell (e.g., a monocyte, amacrophage, a neutrophil or a granulocyte).

In some embodiments of a method disclosed herein, the immune cellpopulation comprises a T cell, e.g., a CD4+ T cell, a CD8+ T cell, a TCRalpha-beta T cell, or a TCR gamma-delta T cell. In some embodiments, a Tcell comprises a memory T cell (e.g., a central memory T cell, or aneffector memory T cell (e.g., a T_(EMRA)) or an effector T cell. In someembodiments, a T cell comprises a tumor infiltrating lymphocyte (TIL).

In some embodiments of a method disclosed herein, the immune cellpopulation is obtained from a healthy subject.

In some embodiments of a method disclosed herein, the immune cellpopulation is obtained from a subject (e.g., from an apheresis samplefrom the subject) having a disease, e.g., a cancer, e.g., as describedherein. In some embodiments, the immune cell population obtained from asubject having a disease, e.g., a cancer, comprises a tumor infiltratinglymphocyte (TIL).

In some embodiments of a method disclosed herein, the method results inan expansion of at least 1.1-10 fold (e.g., at least 1.1, 1.2, 1.3, 1.4,1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold expansion).

In some embodiments of a method disclosed herein, the method furthercomprises contacting the population of cells with an agent thatpromotes, e.g., increases, immune cell expansion. In some embodiments,the agent includes an immune checkpoint inhibitor, e.g., as describedherein. In some embodiments, the agent includes a 4-1BB (CD127) agonist,e.g., an anti-4-1BB antibody.

In some embodiments of a method disclosed herein, the method furthercomprises comprising contacting the population of cells with anon-dividing population of cells, e.g., feeder cells, e.g., irradiatedallogenic human PBMCs.

In some embodiments of a method disclosed herein, an expansion methoddescribed herein comprises expanding the cells for a period of at leastabout 4 hours, 6 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20hours, or 22 hours, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, or for at least about 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks.

In some embodiments of a method disclosed herein, expansion of thepopulation of immune cells, is compared to expansion of a similarpopulation of cells with an antibody that binds to: a CD3 molecule,e.g., CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

In some embodiments of a method disclosed herein, expansion of thepopulation of immune cells, is compared to expansion of a similarpopulation of cells not contacted with the anti-TCRβV antibody molecule.

In some embodiments of a method disclosed herein, expansion of thepopulation of memory effector T cells, e.g., T_(EM) cells, e.g.,T_(EMRA) cells, is compared to expansion of a similar population ofcells with an antibody that binds to: a CD3 molecule, e.g., CD3 epsilon(CD3e) molecule; or a TCR alpha (TCRα) molecule.

In some embodiments of a method disclosed herein, the method results inexpansion of, e.g., selective or preferential expansion of, T cellsexpressing a T cell receptor (TCR) comprising a TCR alpha and/or TCRbeta molecule, e.g., TCR alpha-beta T cells (αβ T cells).

In some embodiments of a method disclosed herein, the method results inexpansion of αβT cells over expansion of T cells expressing a TCRcomprising a TCR gamma and/or TCR delta molecule, e.g., TCR gamma-deltaT cells (γδ T cells). In some embodiments, expansion of αβT cells overγδ T cells results in reduced production of cytokines associated withCRS. In some embodiments, expansion of αβT cells over γδ T cells resultsin immune cells that have reduced capacity to, e.g., are less prone to,induce CRS upon administration into a subject.

In some embodiments of a method disclosed herein, an immune cellpopulation (e.g., T cells (e.g., T_(EMRA) cells or TILs) or NK cells)cultured in the presence of, e.g., expanded with, an anti-TCRβV antibodydisclosed herein does not induce CRS and/or NT when administered into asubject, e.g., a subject having a disease or condition as describedherein.

In some embodiments, the anti-TCRβV antibody molecule in a multispecificmolecule disclosed herein is a first immune cell engager moiety. In someembodiments, the anti-TCRβV antibody molecule does not bind to TCRβ V12,or binds to TCRβ V12 with an affinity and/or binding specificity that isless than (e.g., less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or about 2-, 5-, or 10-fold) the affinity and/or binding specificityof the 16G8 murine antibody or a humanized version thereof as describedin U.S. Pat. No. 5,861,155. In some embodiments, the anti-TCRβV antibodymolecule binds to TCRβ V12 with an affinity and/or binding specificitythat is greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/orbinding specificity of the 16G8 murine antibody or a humanized versionthereof as described in U.S. Pat. No. 5,861,155. In some embodiments,the anti-TCRβV antibody molecule binds to a TCRβV region other than TCRβV12 (e.g., TCRβV region as described herein, e.g., TCRβ V6 subfamily(e.g., TCRβ V6-5*01) with an affinity and/or binding specificity that isgreater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/or bindingspecificity of the 16G8 murine antibody or a humanized version thereofas described in U.S. Pat. No. 5,861,155. In some embodiments, theanti-TCRβV antibody molecule does not comprise the CDRs of the AntibodyB murine antibody.

In some embodiments, the anti-TCRβV antibody molecule in a multispecificmolecule disclosed herein is a first immune cell engager moiety. In someembodiments, the anti-TCRβV antibody molecule does not bind to TCRβV5-5*01 or TCRβ V5-1*01, or binds to TCRβ V5-5*01 or TCRβ V5-1*01 withan affinity and/or binding specificity that is less than (e.g., lessthan about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-,or 10-fold) the affinity and/or binding specificity of the TM23 murineantibody or a humanized version thereof as described in U.S. Pat. No.5,861,155. In some embodiments, the anti-TCRβV antibody molecule bindsto TCRβ V5-5*01 or TCRβ V5-1*01 with an affinity and/or bindingspecificity that is greater than (e.g., greater than about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) theaffinity and/or binding specificity of the TM23 murine antibody or ahumanized version thereof as described in U.S. Pat. No. 5,861,155. Insome embodiments, the anti-TCRβV antibody molecule binds to a TCRβVregion other than TCRβ V5-5*01 or TCRβ V5-1*01 (e.g., TCRβV region asdescribed herein, e.g., TCRβ V6 subfamily (e.g., TCRβ V6-5*01) with anaffinity and/or binding specificity that is greater than (e.g., greaterthan about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-,or 10-fold) the affinity and/or binding specificity of the TM23 murineantibody or a humanized version thereof as described in U.S. Pat. No.5,861,155. In some embodiments, the anti-TCRβV antibody molecule doesnot comprise the CDRs of the TM23 murine antibody.

In some embodiments, the multispecific molecule further comprises asecond immune cell engager moiety. In some embodiments, the first and/orsecond immune cell engager binds to and activates an immune cell, e.g.,an effector cell. In some embodiments, the first and/or second immunecell engager binds to, but does not activate, an immune cell, e.g., aneffector cell. In some embodiments, the second immune cell engager ischosen from an NK cell engager, a T cell engager, a B cell engager, adendritic cell engager, or a macrophage cell engager, or a combinationthereof. In some embodiments, the second immune cell engager comprises aT cell engager which binds to CD3, TCRα, TCRγ, TCRζ, ICOS, CD28, CD27,HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, orCD226.

In some embodiments, a multispecific molecule disclosed herein comprisesa tumor targeting moiety. In some embodiment, the tumor-targeting moietycomprises an antibody molecule (e.g., Fab or scFv), a receptor molecule(e.g., a receptor, a receptor fragment or functional variant thereof),or a ligand molecule (e.g., a ligand, a ligand fragment or functionalvariant thereof), or a combination thereof, that binds to a cancerantigen. In some embodiments, the tumor-targeting moiety binds to acancer antigen present on a cancer, e.g., a hematological cancer, asolid tumor, a metastatic cancer, soft tissue tumor, metastatic lesion,or a combination thereof. In some embodiments, the tumor-targetingmoiety binds to a cancer antigen, e.g., BCMA or FcRH5.

In some embodiments, the tumor-targeting antibody molecule binds to aconformational or a linear epitope on the tumor antigen.

In some embodiments of any of the compositions or methods disclosedherein, the tumor-targeting moiety is an antigen, e.g., a cancerantigen. In some embodiments, the cancer antigen is a tumor antigen orstromal antigen, or a hematological antigen.

In some embodiments of any of the compositions or methods disclosedherein, the tumor-targeting moiety binds to a cancer antigen chosenfrom: BCMA, FcRH5, CD19, CD20, CD22, CD30, CD33, CD38, CD47, CD99,CD123, FcRH5, CLEC12, CD179A, SLAMF7, or NY-ESO1, PDL1, CD47, gangloside2 (GD2), prostate stem cell antigen (PSCA), prostate specific membraneantigen (PMSA), prostate-specific antigen (PSA), carcinoembryonicantigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72,BING-4, Calcium-activated chloride channel 2, Cyclin-B1, 9D7, Ep-CAM,EphA3, Her2/neu, Telomerase, SAP-1, Survivin, NY-ESO-1/LAGE-1, PRAME,SSX-2, Melan-A/MART-1, Gp100/pme117, Tyrosinase, TRP-1/-2, MC1R,β-catenin, BRCA1/2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor,AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, 13-catenin, CDK4,CDC27, a actinin-4, TRP1/gp75, TRP2, gp100, Melan-A/MART1, gangliosides,WT1, EphA3, Epidermal growth factor receptor (EGFR), MART-2, MART-1,MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, RCC, RUI1, RUI2,SAGE, TRG, TRP1, TSTA, Folate receptor alpha, L1-CAM, CAIX, gpA33, GD3,GM2, VEGFR, Intergrins (Integrin alphaVbeta3, Integrin alpha5Beta1),Carbohydrates (Le), IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, (FAP),TGF-beta, hyaluronic acid, collagen, e.g., collagen IV, tenascin C, ortenascin W.

In some embodiments of any of the compositions or methods disclosedherein, the cancer is a solid tumor including but not limited to:pancreatic (e.g., pancreatic adenocarcinoma) cancer, breast cancer,colorectal cancer, lung cancer (e.g., small or non-small cell lungcancer), skin cancer, ovarian cancer, or liver cancer.

In some embodiments of any of the compositions or methods disclosedherein, the cancer antigen or tumor antigen is a hematological antigen.In some embodiments, the cancer or tumor antigen is chosen from one ormore of: BCMA, FcRH5, CD19, CD20, CD22, CD30, CD33, CD38, CD47, CD99,CD123, FcRH5, CLEC12, CD179A, SLAMF7, or NY-ESO1. In some embodiments,the tumor-targeting moiety binds to one or both of BCMA or FcRH5.

In some embodiments, the tumor-targeting moiety binds to BCMA. Inembodiments, the tumor-targeting moiety comprises a BCMA targetingmoiety. In some embodiments, the tumor-targeting moiety comprising aBCMA targeting moiety binds to a BCMA antigen on the surface of a cell,e.g., a cancer or hematopoietic cell. The BCMA antigen can be present ona primary tumor cell, or a metastatic lesion thereof. In someembodiments, the cancer is a hematological cancer, e.g., multiplemyeloma. For example, the BCMA antigen can be present on a tumor, e.g.,a tumor of a class typified by having one or more of: limited tumorperfusion, compressed blood vessels, or fibrotic tumor interstitium. Insome embodiments, the tumor targeting moiety comprising a BCMA targetingmoiety comprises an anti-BCMA antibody or antigen-binding fragmentthereof described in U.S. Pat. Nos. 8,920,776, 9,243,058, 9,340,621,8,846,042, 7,083,785, 9,545,086, 7,276,241, 9,034,324, 7,799,902,9,387,237, 8,821,883, US861745, US20130273055, US20160176973,US20150368351, US20150376287, US20170022284, US20160015749,US20140242077, US20170037128, US20170051068, US20160368988,US20160311915, US20160131654, US20120213768, US20110177093,US20160297885, EP3137500, EP2699259, EP2982694, EP3029068, EP3023437,WO2016090327, WO2017021450, WO2016110584, WO2016118641, WO2016168149,the entire contents of which are incorporated herein by reference.

In some embodiments, the BCMA-targeting moiety includes an antibodymolecule (e.g., Fab or scFv) that binds to BCMA. In some embodiments,the antibody molecule to BCMA comprises one, two, or three CDRs from anyof the heavy chain variable domain sequences of Table 14, or a closelyrelated CDR, e.g., CDRs which have at least one amino acid alteration,but not more than two, three or four alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions) from any ofthe CDR sequences of Table 14. In some embodiments, the antibodymolecule to BCMA comprises a heavy chain variable domain sequence chosenfrom any of the amino acid sequences of Table 14, or an amino acidsequence substantially identical thereto (e.g., 95% to 99.9% identicalthereto, or having at least one amino acid alteration, but not more thanfive, ten or fifteen alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions)).

In some embodiments, the tumor-targeting moiety binds to FcRH5. Inembodiments, the tumor-targeting moiety comprises a FcRH5 targetingmoiety. In some embodiments, the tumor-targeting moiety comprising aFcRH5 targeting moiety binds to a FcRH5 antigen on the surface of acell, e.g., a cancer or hematopoietic cell. The FcRH5 antigen can bepresent on a primary tumor cell, or a metastatic lesion thereof. In someembodiments, the cancer is a hematological cancer, e.g., multiplemyeloma. For example, the FcRH5 antigen can be present on a tumor, e.g.,a tumor of a class typified by having one or more of: limited tumorperfusion, compressed blood vessels, or fibrotic tumor interstitium. Insome embodiments, the tumor targeting moiety comprising a FcRH5targeting moiety comprises an anti-FcRH5 antibody or antigen-bindingfragment thereof described in U.S. Pat. No. 7,999,077 the entirecontents of which are incorporated herein by reference.

In some embodiments of any of the compositions or methods disclosedherein, the cancer is a hematological cancer including, but not limitedto: a B-cell or T cell malignancy, e.g., Hodgkin's lymphoma,Non-Hodgkin's lymphoma (e.g., B cell lymphoma, diffuse large B celllymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle celllymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma,lymphoplasmacytic lymphoma, hairy cell leukemia), acute myeloid leukemia(AML), chronic myeloid leukemia, myelodysplastic syndrome, multiplemyeloma, and acute lymphocytic leukemia. In some embodiments, thehematological cancer is multiple myeloma.

In some embodiments, a multispecific molecule disclosed herein furthercomprises a cytokine molecule, e.g., one or two cytokine molecules. Insome embodiments, the cytokine molecule is chosen from interleukin-2(IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15(IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or interferongamma, or a fragment, variant or combination thereof. In someembodiments, is a monomer or a dimer. In some embodiments, the cytokinemolecule further comprises a receptor dimerizing domain, e.g., anIL15Ralpha dimerizing domain. In some embodiments, the cytokine molecule(e.g., IL-15) and the receptor dimerizing domain (e.g., an IL15Ralphadimerizing domain) are not covalently linked, e.g., are non-covalentlyassociated.

In some embodiments, a multispecific molecule disclosed hereincomprises:

(i) an anti-TCRβV antibody molecule (e.g., an anti-TCRβV antibodymolecule as described herein); and(ii) a tumor-targeting antibody molecule (e.g., an antibody moleculethat binds to a hematological antigen as described herein, e.g., chosenfrom one or more of BCMA, FcRH5, CD19, CD22, CD33, CD123, FcRH5, CD179a,or CLEC12).

In some embodiments, the multispecific molecule disclosed hereincomprises the anti-TCRβV antibody molecule of (i), the tumor-targetingantibody molecule of (ii) and a cytokine molecule as described herein,e.g., an IL-12 cytokine molecule.

In some embodiments, the multispecific molecule comprises an anti-TCRβVantibody molecule as described herein; and a tumor-targeting antibodymolecule that binds to one or both of BCMA or FcRH5. In someembodiments, the multispecific molecule further comprises an IL-12cytokine molecule. The multispecific molecule can be used to treat aBCMA- or FcRH5-expressing hematological cancer, e.g., multiple myeloma.

In some embodiments, the multispecific molecule comprises an anti-TCRβVantibody molecule as described herein; and a tumor-targeting antibodymolecule that binds one or more of CD19, CD22, or CD123. In someembodiments, the multispecific molecule further comprises an IL-12cytokine molecule. The multispecific molecule can be used to treat aCD19-, CD22-, or CD123-expressing hematological cancer, e.g., leukemiaor lymphoma. In some embodiments, the CD19-, CD22-, or CD123-expressinghematological cancer is chosen from a B-cell or T cell malignancy, e.g.,Hodgkin's lymphoma, Non-Hodgkin's lymphoma (e.g., B cell lymphoma,diffuse large B cell lymphoma, follicular lymphoma, chronic lymphocyticleukemia, mantle cell lymphoma, marginal zone B-cell lymphoma, Burkittlymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia), acutemyeloid leukemia (AML), chronic myeloid leukemia, myelodysplasticsyndrome, multiple myeloma, and acute lymphocytic leukemia. In someembodiments, the hematological cancer is multiple myeloma.

In some embodiments, a multispecific molecule disclosed herein furthercomprises an immunoglobulin constant region (e.g., Fc region) chosenfrom the heavy chain constant regions of IgG1, IgG2, and IgG4, moreparticularly, the heavy chain constant region of human IgG1, IgG2 orIgG4. In some embodiments, the immunoglobulin constant region (e.g., anFc region) is linked, e.g., covalently linked to, one or more oftumor-targeting moiety, the immune cell engager, the cytokine molecule,or the stromal modifying moiety. In some embodiments, an interface of afirst and second immunoglobulin chain constant regions (e.g., Fc region)is altered, e.g., mutated, to increase or decrease dimerization, e.g.,relative to a non-engineered interface. In some embodiments, thedimerization of the immunoglobulin chain constant region (e.g., Fcregion) is enhanced by providing an Fc interface of a first and a secondFc region with one or more of: a paired cavity-protuberance (“knob-in-ahole”), an electrostatic interaction, or a strand-exchange, such that agreater ratio of heteromultimer:homomultimer forms, e.g., relative to anon-engineered interface. In some embodiments,

In some embodiments, a multispecific molecule disclosed herein furthercomprises a linker, e.g., a linker described herein, optionally whereinthe linker is selected from: a cleavable linker, a non-cleavable linker,a peptide linker, a flexible linker, a rigid linker, a helical linker,or a non-helical linker.

In some embodiments, the multispecific molecule comprises at least twonon-contiguous polypeptide chains.

In some embodiments, the multispecific molecule comprises the followingconfiguration:

-   -   A, B-[dimerization module]-C, -D        wherein:        (1) the dimerization module comprises an immunoglobulin constant        domain, e.g., a heavy chain constant domain (e.g., a homodimeric        or heterodimeric heavy chain constant region, e.g., an Fc        region), or a constant domain of an immunoglobulin variable        region (e.g., a Fab region); and        (2) A, B, C, and D are independently absent; (i) an antigen        binding domain that preferentially binds to a first immune cell        engager comprising an anti-TCRβV antibody molecule disclosed        herein; (ii) a tumor targeting moiety (e.g., a tumor-targeting        antibody molecule as described herein), (iii) a second immune        cell engager chosen from a T cell engager, an NK cell engager, a        B cell engager, a dendritic cell engager, or a macrophage cell        engager; (iv) a cytokine molecule; or (v) a stromal modifying        moiety, provided that:        at least one, two, or three of A, B, C, and D comprises an        antigen binding domain that preferentially binds to a TCRβV        region disclosed herein, and        any of the remaining A, B, C, and D is absent or comprises one        of a tumor targeting moiety, a second immune cell engager, a        cytokine molecule, or a stromal modifying moiety.

In some embodiments, the dimerization module comprises one or moreimmunoglobulin chain constant regions (e.g., Fc regions) comprising oneor more of: a paired cavity-protuberance (“knob-in-a hole”), anelectrostatic interaction, or a strand-exchange. In some embodiments,the one or more immunoglobulin chain constant regions (e.g., Fc regions)comprise an amino acid substitution at a position chosen from one ormore of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399,405, 407, or 409, e.g., of the Fc region of human IgG1. In someembodiments, the one or more immunoglobulin chain constant regions(e.g., Fc regions) comprise an amino acid substitution chosen from:T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), orT366W (e.g., corresponding to a protuberance or knob), or a combinationthereof.

In some embodiments, the multispecific molecule further comprises alinker, e.g., a linker between one or more of: the antigen bindingdomain of an anti-TCRβV antibody molecule disclosed herein and the tumortargeting moiety; the antigen binding domain of an anti-TCRβV antibodymolecule disclosed herein and the second immune cell engager, theantigen binding domain of an anti-TCRβV antibody molecule disclosedherein and the cytokine molecule, the antigen binding domain of ananti-TCRβV antibody molecule disclosed herein and the stromal modifyingmoiety, the second immune cell engager and the cytokine molecule, thesecond immune cell engager and the stromal modifying moiety, thecytokine molecule and the stromal modifying moiety, the antigen bindingdomain of an anti-TCRβV antibody molecule disclosed herein and thedimerization module, the second immune cell engager and the dimerizationmodule, the cytokine molecule and the dimerization module, the stromalmodifying moiety and the dimerization module, the tumor targeting moietyand the dimerization module, the tumor targeting moiety and the cytokinemolecule, the tumor targeting moiety and the second immune cell engager,or the tumor targeting moiety and the antigen binding domain of ananti-TCRβV antibody molecule disclosed herein. In some embodiments, thelinker is chosen from: a cleavable linker, a non-cleavable linker, apeptide linker, a flexible linker, a rigid linker, a helical linker, ora non-helical linker. In some embodiments, the linker is a peptidelinker. In some embodiments, the peptide linker comprises Gly and Ser.In some embodiments, the peptide linker comprises an amino acid sequencechosen from SEQ ID NOs: 142-145 or 175-178.

In some embodiments of a method or composition for use disclosed herein,the disease is a cancer chosen from: a hematological cancer, a solidtumor, a metastatic cancer, soft tissue tumor, metastatic lesion, or acombination thereof.

In some embodiments of a method or composition for use disclosed herein,the cancer is a solid tumor chosen from: a melanoma, a pancreatic cancer(e.g., pancreatic adenocarcinoma), a breast cancer, a colorectal cancer(CRC), a lung cancer (e.g., small or non-small cell lung cancer), a skincancer, an ovarian cancer, or a liver cancer. In some embodiments, thecancer is melanoma or CRC.

In some embodiments of a method or composition for use disclosed hereinthe cancer is a hematological cancer chosen from: a B-cell or T cellmalignancy, e.g., Hodgkin's lymphoma, Non-Hodgkin's lymphoma (e.g., Bcell lymphoma, diffuse large B cell lymphoma, follicular lymphoma,chronic lymphocytic leukemia, mantle cell lymphoma, marginal zone B-celllymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cellleukemia), acute myeloid leukemia (AML), chronic myeloid leukemia,myelodysplastic syndrome, multiple myeloma, or acute lymphocyticleukemia. In some embodiments, the hematological cancer is multiplemyeloma. In some embodiments, the hematological cancer is CLL or DLBCL.

In some embodiments of a method or composition for use disclosed hereinthe sample from the subject comprises a blood sample, e.g., a peripheralblood sample, a biopsy, e.g., a tumor biopsy, or a bone marrow sample.IN some embodiments, the sample comprises a biological sample comprisingimmune effector cells, e.g., T cells, or NK cells. In some embodiments,T cells comprise a CD4 T cell, a CD8 T cell, (e.g., an effector T cellor a memory T cell (e.g., a memory effector T cell (e.g., T_(EM) cell,e.g., T_(EMRA) cell), or a tumor infiltrating lymphocyte (TIL).

What is claimed is:
 1. A method of expanding T cells that expresses a Tcell receptor beta variable region (TCRβV) in a T cell population, themethod comprising: contacting the T cell population with a compositioncomprising a multispecific molecule, wherein the multispecific moleculecomprises a first domain that binds to a first target molecule and asecond domain that binds to a second target molecule, wherein the firsttarget molecule is a TCRβV and the second target molecule is a targetmolecule on a target cell that is different from the first targetmolecule, and wherein the first domain contacts the TCRβV of a T cellreceptor (TCR) expressed by the T cells in the T cell population,thereby expanding the T cells in the T cell population.
 2. The method ofclaim 1, wherein the T cell population is an in vivo T cell population.3. The method of claim 1, wherein the second domain comprises atumor-targeting domain, a cytokine molecule, or a stromal modifyingdomain.
 4. The method of claim 1, wherein the multispecific moleculecomprises at least two non-contiguous polypeptide chains, wherein afirst polypeptide chain of the at least two non-contiguous polypeptidechains comprises a first member of a dimerization module, and a secondpolypeptide chain of the at least two non-contiguous polypeptide chainscomprises a second member of the dimerization module, wherein the firstpolypeptide chain and the second polypeptide chain form a complex viathe first member of the dimerization module and the second member of thedimerization module.
 5. The method of claim 4, wherein the firstpolypeptide chain comprises the first domain and the second polypeptidechain comprises the second domain, wherein: (i) the first polypeptidechain comprises the first domain linked to the first member of thedimerization module, and the second polypeptide chain comprises thesecond domain linked to the second member of the dimerization module;(ii) the first polypeptide chain comprises a first portion of the firstdomain linked to the first member of the dimerization module, and thesecond polypeptide chain comprises a first portion of the second domainlinked to the second member of the dimerization module; wherein the atleast two non-contiguous polypeptide chains comprises a thirdpolypeptide chain comprising a second portion of the first domain and afourth polypeptide chain comprising a second portion of the seconddomain; (iii) the first polypeptide chain comprises a first portion ofthe first domain linked to the first member of the dimerization module,and the second polypeptide chain comprises the second domain linked tothe second member of the dimerization module; wherein the at least twonon-contiguous polypeptide chains comprises a third polypeptide chaincomprising a second portion of the first domain; or (iv) the firstpolypeptide chain comprises the first domain linked to the first memberof the dimerization module, and the second polypeptide chain comprises afirst portion of the second domain linked to the second member of thedimerization module; wherein the at least two non-contiguous polypeptidechains comprises a third polypeptide chain comprising a second portionof the second domain.
 6. The method of claim 5, wherein themultispecific molecule further comprises a linker between the firstdomain and the first member of the dimerization module, a linker betweenthe second domain and the second member of the dimerization module, alinker between the first portion of the first domain and the firstmember of the dimerization module, a linker between the first portion ofthe second domain and the second member of the dimerization module, alinker between the first member of the dimerization module and thesecond domain, a linker between the first member of the dimerizationmodule and the first portion of the second domain or a combinationthereof, wherein the linker is selected from a cleavable linker, anon-cleavable linker, a peptide linker, a flexible linker, a rigidlinker, a helical linker, and a non helical linker.
 7. The method ofclaim 4, wherein the first polypeptide chain comprises the first domainand the second domain, wherein the first polypeptide chain comprises:(i) the first domain linked to the first member of the dimerizationmodule linked to the second domain; (ii) a first portion of the firstdomain linked to the first member of the dimerization module linked to afirst portion of the second domain, wherein the at least twonon-contiguous polypeptide chains comprises a third polypeptide chaincomprising a second portion of the first domain and a fourth polypeptidechain comprising a second portion of the second domain; (iii) a firstportion of the first domain linked to the first member of thedimerization module linked to the second domain, wherein the at leasttwo non-contiguous polypeptide chains comprises a third polypeptidechain comprising a second portion of the first domain; or (iv) the firstdomain linked to the first member of the dimerization module linked to afirst portion of the second domain, wherein the at least twonon-contiguous polypeptide chains comprises a third polypeptide chaincomprising a second portion of the second domain.
 8. The method of claim7, wherein the multispecific molecule further comprises a linker betweenthe first domain and the first member of the dimerization module, alinker between the second domain and the second member of thedimerization module, a linker between the first portion of the firstdomain and the first member of the dimerization module, a linker betweenthe first portion of the second domain and the second member of thedimerization module, a linker between the first member of thedimerization module and the second domain, a linker between the firstmember of the dimerization module and the first portion of the seconddomain or a combination thereof, wherein the linker is selected from acleavable linker, a non-cleavable linker, a peptide linker, a flexiblelinker, a rigid linker, a helical linker, and a non helical linker. 9.The method of claim 1, wherein the multispecific molecule comprises apolypeptide sequence comprising: (i) the first domain linked to thesecond domain; (ii) a first portion of the first domain linked to afirst portion of the second domain, wherein the polypeptide sequencefurther comprises a second portion of the first domain and a secondportion of the second domain; (iii) a first portion of the first domainlinked to the second domain, wherein the polypeptide sequence furthercomprises a second portion of the first domain; or (iv) the first domainlinked to a first portion of the second domain, wherein the polypeptidesequence further comprises a second portion of the second domain. 10.The method of claim 9, wherein the polypeptide sequence furthercomprises a linker between the first domain and the second domain, alinker between the first portion of the first domain and the firstportion of the second domain, a linker between the first portion of thefirst domain and the second domain, a linker between the first domainand the first portion of the second domain, or a combination thereof,wherein the linker is selected from a cleavable linker, a non-cleavablelinker, a peptide linker, a flexible linker, a rigid linker, a helicallinker, and a non-helical linker.
 11. The method of claim 1, wherein theTCRβV is TCRβV1, TCRβV2, TCRβV3, TCRβV4, TCRβV5, TCRβV6, TCRβV7, TCRβV8,TCRβV9, TCRβV10, TCRβV11, TCRβV12, TCRβV19, TCRβV20, TCRβV21, TCRβV23,TCRβV24, TCRβV25, TCRβV26, TCRβV27, TCRβV28, TCRβV29 or TCRβV30.
 12. Themethod of claim 1, wherein the TCRβV is TCRβV2, TCRβV4-1, TCRβV4-2,TCRβV5-1, TCRβV5-5, TCRβV5-6, TCRβV6, TCRβV6-5, TCRβV6-6, TCRβV6-9,TCRβV7-2, TCRβV7-3, TCRβV7-8, TCRβV7-9, TCRβV9, TCRβV10-1, TCRβV10-2,TCRβV10-3, TCRβV11-2, TCRβV12-3, TCRβV12-4, TCRβV12-5, TCRβV19,TCRβV20-1, TCRβV21, TCRβV24-1, TCRβV25-1 or TCRβV28.
 13. The method ofclaim 1, wherein the TCRβV is TCRβV2, TCRβV3-1, TCRβV4-1, TCRβV4-2,TCRβ3V5-1, TCRβV5-4, TCRβV5-5, TCRβV5-6, TCRβV6-1, TCRβV6-5, TCRβV6-6,TCRβV7-3, TCRβV7-6, TCRβV7-8, TCRβV9, TCRβV11-2, TCRβV19, TCRβV20-1,TCRβV24-1, TCRβV27, TCRβV28, TCRβV29-1 or TCRβV30.
 14. The method ofclaim 1, wherein second target molecule is selected from the groupconsisting of BCMA, FcRH5, CD19, CD20, CD22, CD30, CD33, CD38, CD47,CD99, CD123, FcRH5, CLEC12, CD179A, SLAMF7, or NY-ESO1, PDL1, CD47,gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specificmembrane antigen (PSMA), prostate-specific antigen (PSA),carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature lamininreceptor, TAG-72, BING-4, Calcium-activated chloride channel 2,Cyclin-B1, 9D7, Ep-CAM, EphA3, Her2/neu, Telomerase, SAP-1, Survivin,NY-ESO-1/LAGE-1, PRAME, SSX-2, Melan-A/MART-1, Gp100/pme117, Tyrosinase,TRP-1/-2, MC1R, b-catenin, BRCA1/2, CDK4, CML66, Fibronectin, p53, Ras,TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1,b-catenin, CDK4, CDC27, a actinin-4, TRP1/gp75, TRP2, gp100,Melan-A/MART1, gangliosides, WT1, EphA3, Epidermal growth factorreceptor (EGFR), MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1,NPM, OA1, OGT, RCC, RUI1, RUI2, SAGE, TRG, TRP1, TSTA, Folate receptoralpha, L1-CAM, CAIX, gpA33, GD3, GM2, VEGFR, Intergrin, a carbohydrates,IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, TGF-beta, hyaluronic acid,collagen, tenascin C and tenascin W.
 15. The method of claim 1, whereinthe second domain is an NK cell engager, a T cell engager, a B cellengager, a dendritic cell engager, or a macrophage cell engager.
 16. Themethod of claim 15, wherein the second domain is a T cell engager andwherein the second target molecule is a TCRβV other than the TCRβV towhich the first domain binds.
 17. The method of claim 15, wherein thesecond target molecule is not a TCRβV.
 18. The method of claim 15,wherein the second target molecule is CD19.
 19. The method of claim 15,wherein the second target molecule is CD3.
 20. The method of claim 15,wherein the second target molecule is CD123.
 21. The method of claim 3,wherein the second domain comprises a tumor-targeting domain and thesecond target molecule is a cancer antigen.
 22. The method of claim 21,wherein the cancer antigen is a hematological cancer antigen, a solidtumor antigen, a metastatic cancer antigen, a soft tissue tumor antigen,a cancer antigen of a metastatic lesion or a stromal antigen.
 23. Themethod of claim 22, wherein the cancer antigen is: (i) the solid tumorantigen, wherein the solid tumor is pancreatic cancer, breast cancer,colorectal cancer, lung cancer, skin cancer, ovarian cancer, or livercancer; or (ii) the hematological cancer antigen, wherein thehematological cancer is a B-cell malignancy or a T cell malignancy. 24.The method of claim 22, wherein the cancer antigen is the hematologicalcancer antigen and the B-cell malignancy or the T cell malignancy isHodgkin's lymphoma, Non-Hodgkin's lymphoma, acute myeloid leukemia(AML), chronic myeloid leukemia, myelodysplastic syndrome, multiplemyeloma, or acute lymphocytic leukemia.
 25. The method of claim 23,wherein the cancer antigen is the hematological cancer antigen and theB-cell malignancy is Hodgkin's lymphoma, wherein the Non-Hodgkin'slymphoma is B cell lymphoma, diffuse large B cell lymphoma, follicularlymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, marginalzone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, orhairy cell leukemia.
 26. The method of claim 3, wherein the seconddomain comprises a cytokine molecule selected from the group consistingof interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12),interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21),interferon gamma and functional fragments or variants thereof.
 27. Themethod of claim 1, wherein binding of the first domain to the TCRβV andbinding of the second molecule to the target molecule promotes the Tcells to kill cancer cells.
 28. The method of claim 1, wherein thetarget cell is a T cell.
 29. The method of claim 1, wherein the targetcell is a non-cancer cell.
 30. The method of claim 1, wherein the methodexpands T cells in vivo.